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Engineered dual selection for directed evolution of SpCas9 PAM specificity

Author

Listed:
  • Gregory W. Goldberg

    (NYU Langone Health)

  • Jeffrey M. Spencer

    (NYU Langone Health)

  • David O. Giganti

    (NYU Langone Health)

  • Brendan R. Camellato

    (NYU Langone Health)

  • Neta Agmon

    (NYU Langone Health
    Neochromosome, Inc., Alexandria Center for Life Science)

  • David M. Ichikawa

    (NYU Langone Health)

  • Jef D. Boeke

    (NYU Langone Health
    NYU Tandon School of Engineering)

  • Marcus B. Noyes

    (NYU Langone Health)

Abstract

The widely used Streptococcus pyogenes Cas9 (SpCas9) nuclease derives its DNA targeting specificity from protein-DNA contacts with protospacer adjacent motif (PAM) sequences, in addition to base-pairing interactions between its guide RNA and target DNA. Previous reports have established that the PAM specificity of SpCas9 can be altered via positive selection procedures for directed evolution or other protein engineering strategies. Here we exploit in vivo directed evolution systems that incorporate simultaneous positive and negative selection to evolve SpCas9 variants with commensurate or improved activity on NAG PAMs relative to wild type and reduced activity on NGG PAMs, particularly YGG PAMs. We also show that the PAM preferences of available evolutionary intermediates effectively determine whether similar counterselection PAMs elicit different selection stringencies, and demonstrate that negative selection can be specifically increased in a yeast selection system through the fusion of compensatory zinc fingers to SpCas9.

Suggested Citation

  • Gregory W. Goldberg & Jeffrey M. Spencer & David O. Giganti & Brendan R. Camellato & Neta Agmon & David M. Ichikawa & Jef D. Boeke & Marcus B. Noyes, 2021. "Engineered dual selection for directed evolution of SpCas9 PAM specificity," Nature Communications, Nature, vol. 12(1), pages 1-16, December.
    • Handle: RePEc:nat:natcom:v:12:y:2021:i:1:d:10.1038_s41467-020-20650-x
    DOI: 10.1038/s41467-020-20650-x
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    Cited by:

    1. Jian Wang & Yuxi Teng & Ruihua Zhang & Yifei Wu & Lei Lou & Yusong Zou & Michelle Li & Zhong-Ru Xie & Yajun Yan, 2021. "Engineering a PAM-flexible SpdCas9 variant as a universal gene repressor," Nature Communications, Nature, vol. 12(1), pages 1-10, December.

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