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Protocols for C-Brick DNA Standard Assembly Using Cpf1
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Programmable sequential mutagenesis by inducible Cpf1 crRNA array inversion.

Ryan D Chow1,2,3, Hyunu Ray Kim1,2, Sidi Chen4,5,6,7,8,9,10

  • 1Department of Genetics, Yale University School of Medicine, New Haven, CT, 06511, USA.

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Researchers developed Cpf1-Flip, a novel system for sequential mutagenesis. This tool precisely models the stepwise genetic changes crucial for understanding cancer evolution and immunotherapy resistance.

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Area of Science:

  • Genetics and Genomics
  • Cancer Biology
  • Molecular Biology

Background:

  • Genetic alterations accumulate sequentially in biological processes like development, evolution, and cancer.
  • Specific phenotypes arise from precise temporal sequences of genetic events.
  • Existing methods for modeling cancer mutations often fail to capture this stepwise progression.

Purpose of the Study:

  • To introduce Cpf1-Flip, a flexible system for inducible sequential mutagenesis.
  • To demonstrate the utility of Cpf1-Flip in modeling cancer evolution and immunotherapy resistance.

Main Methods:

  • Development of the Cpf1-Flip system utilizing inducible inversion of a crRNA array (FlipArray).
  • Application of Cpf1-Flip for stepwise mutagenesis in murine and human cell lines.
  • Utilizing Cpf1-Flip in a pooled-library approach to study resistance mutations in cancer immunotherapy.

Main Results:

  • Cpf1-Flip enables controlled, sequential introduction of multiple mutations.
  • The system was successfully applied in both murine and human cells.
  • Proof-of-concept demonstrated modeling of diverse cancer immunotherapy resistance mutations.

Conclusions:

  • Cpf1-Flip provides a versatile and controlled method for precise sequential mutagenesis.
  • This system facilitates the study of complex genetic alterations in various biological contexts.
  • Cpf1-Flip is a valuable tool for modeling cancer progression and therapeutic resistance.