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Genome Engineering of Primary Human B Cells Using CRISPR/Cas9
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Highly parallel genome variant engineering with CRISPR-Cas9.

Meru J Sadhu1,2,3,4, Joshua S Bloom5,6,7,8, Laura Day9,10,11

  • 1Department of Human Genetics, University of California, Los Angeles, Los Angeles, CA, USA. msadhu@mednet.ucla.edu.

Nature Genetics
|April 11, 2018
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Summary
This summary is machine-generated.

This study introduces a CRISPR-based method for precise genome-wide variant engineering. The approach revealed that premature-termination codons are usually harmful, with some essential genes proving dispensable.

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Area of Science:

  • Genetics
  • Molecular Biology
  • Biotechnology

Background:

  • Understanding DNA variant effects is crucial for biology and medicine.
  • High-throughput measurement of these effects remains a challenge.
  • CRISPR-Cas9 enables targeted DNA double-strand breaks for genetic studies.

Purpose of the Study:

  • To develop a CRISPR-library-based method for high-throughput genome-wide variant engineering.
  • To investigate the functional consequences of premature-termination codons (PTCs) in essential yeast genes.
  • To identify essential genes that may be dispensable or possess dispensable regions.

Main Methods:

  • Utilized a CRISPR-library approach for precise genome-wide variant engineering.
  • Generated and analyzed premature-termination codons across all annotated essential genes in yeast.
  • Assessed the impact of PTC location on gene function and protein domains.

Main Results:

  • Most premature-termination codons were deleterious, particularly when not near the 3' end or affecting protein domains.
  • Discovered that some genes considered essential are, in fact, dispensable.
  • Identified significant dispensable regions within other supposedly essential genes.

Conclusions:

  • The developed CRISPR-based method allows for efficient and precise genome-wide variant profiling.
  • PTC tolerance is context-dependent, influenced by location and protein domain integrity.
  • The study redefines gene essentiality and highlights the plasticity of essential gene regions.