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Gregory W Goldberg1, Jeffrey M Spencer2, David O Giganti2

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|January 14, 2021
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Researchers engineered Streptococcus pyogenes Cas9 (SpCas9) variants with altered DNA targeting specificity. The evolved SpCas9 shows improved activity on NAG protospacer adjacent motif (PAM) sequences and reduced activity on NGG PAMs.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Gene Editing

Background:

  • Streptococcus pyogenes Cas9 (SpCas9) is a widely used gene editing tool.
  • Its DNA targeting specificity relies on guide RNA base-pairing and protospacer adjacent motif (PAM) recognition.
  • Altering SpCas9 PAM specificity is crucial for expanding its applications.

Purpose of the Study:

  • To evolve SpCas9 variants with altered PAM specificities using in vivo directed evolution.
  • To achieve improved activity on NAG PAMs and reduced activity on NGG PAMs.
  • To investigate the role of selection stringency in shaping PAM preferences.

Main Methods:

  • Utilized in vivo directed evolution systems with simultaneous positive and negative selection.
  • Employed protein engineering strategies to modify SpCas9.
  • Incorporated compensatory zinc finger fusions to enhance negative selection in yeast.

Main Results:

  • Developed SpCas9 variants with commensurate or improved activity on NAG PAMs compared to wild type.
  • Achieved reduced activity on NGG PAMs, especially YGG PAMs, in evolved variants.
  • Demonstrated that evolutionary intermediates influence counterselection PAM stringency.
  • Showed that negative selection can be specifically increased via zinc finger fusion.

Conclusions:

  • Successfully evolved SpCas9 variants with shifted PAM specificities.
  • Highlighted the effectiveness of simultaneous positive and negative selection for engineering Cas9 nucleases.
  • Provided insights into controlling gene editing specificity through protein engineering.