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CRISPR/Cas9 Genome Editing01:28

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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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Genome Engineering of Primary Human B Cells Using CRISPR/Cas9
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Highly multiplexed genome engineering using CRISPR/Cas9 gRNA arrays.

Morito Kurata1,2,3, Natalie K Wolf1,4, Walker S Lahr1,2,4

  • 1Masonic Cancer Center, University of Minnesota, Minneapolis, MN, United States of America.

Plos One
|September 18, 2018
PubMed
Summary
This summary is machine-generated.

We developed a novel CRISPR method using golden gate assembly to create single guide RNA arrays for multiplex genome engineering. This system efficiently delivers multiple guide RNAs, enhancing CRISPR applications in research and therapeutics.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • The CRISPR/Cas9 system is a powerful tool for genome engineering, widely used in research and therapeutics.
  • Current methods for multiplex genome engineering using CRISPR/Cas9 often involve delivering multiple plasmids, which can be inefficient.
  • Delivering multiple guide RNAs (gRNAs) is crucial for complex genetic modifications and modeling biological processes.

Purpose of the Study:

  • To develop an efficient method for multiplex genome engineering using CRISPR/Cas9.
  • To overcome the limitations of delivering multiple plasmids for multiplexed CRISPR applications.
  • To create a single transcript encoding multiple gRNAs for enhanced delivery and efficiency.

Main Methods:

  • Developed a golden gate assembly method to link multiple gRNAs using Csy4 ribonuclease sequences.
  • Constructed a single gRNA array transcript containing up to 10 gRNAs.
  • Optimized the expression of the gRNA array under a strong pol II promoter.

Main Results:

  • Successfully assembled and delivered a single transcript encoding a gRNA array with up to 10 gRNAs.
  • Demonstrated efficient multiplex genome engineering using the developed gRNA array system.
  • Showcased the system's compatibility with existing CRISPR applications.

Conclusions:

  • The novel golden gate-based gRNA array system significantly enhances the capability and accessibility of multiplex genome engineering.
  • This method provides an efficient alternative to delivering multiple plasmids for complex CRISPR applications.
  • The developed system holds promise for advancing research and therapeutic applications requiring precise manipulation of multiple genomic loci.