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Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage

Péter István Kulcsár1,2,3, András Tálas1,4, Krisztina Huszár1,2,5

  • 1Institute of Enzymology, Research Centre for Natural Sciences of the Hungarian Academy of Sciences, Budapest, Hungary.

Genome Biology
|October 8, 2017
PubMed
Abstract

Insights

Highly enhanced fidelity Streptococcus pyogenes Cas9 (SpCas9) variants (HeFSpCas9s) improve specificity for challenging targets. Optimal nuclease selection is crucial for precise gene editing applications sensitive to off-target effects.

Area of Science:

  • Molecular Biology
  • Biotechnology
  • Genetics

Background:

  • Engineered variants like eSpCas9 and SpCas9-HF1 reduced off-target activity of Streptococcus pyogenes Cas9 (SpCas9).
  • A subset of targets still exhibit significant off-target effects even with high-fidelity SpCas9 variants.
  • New Highly enhanced Fidelity SpCas9 (HeFSpCas9) variants were developed by combining mutations from eSpCas9 and SpCas9-HF1.

Purpose of the Study:

  • To evaluate HeFSpCas9 variants for improved specificity in gene editing.
  • To identify factors influencing nuclease specificity and determine optimal variants for sensitive applications.
  • To compare the performance of HeFSpCas9s against existing high-fidelity SpCas9 variants.

Main Methods:

  • Generation of HeFSpCas9 nuclease variants incorporating mutations from eSpCas9 and SpCas9-HF1.
  • Side-by-side examination of engineered nuclease variants.
  • Analysis of target cleavability and off-target effects.
  • Assessment of DNA-binding and cleavage activities of SpCas9 variants.

Main Results:

  • HeFSpCas9 variants demonstrate substantially improved specificity for targets problematic for eSpCas9 and SpCas9-HF1.
  • These high-fidelity nucleases generally require perfectly matching 20-nucleotide spacers for optimal activity.
  • Mutations in HeFSpCas9 variants can reduce cleavage but not DNA-binding affinity.
  • Targets can be ranked based on cleavability and off-target effects with these nucleases.

Conclusions:

  • No single high-fidelity nuclease variant is universally superior; optimal choice depends on the specific target.
  • A framework is provided for developing new nuclease variants for challenging targets.
  • A method is offered for identifying the best nuclease for a given target without predictive tools.
Keywords:
CRISPRCas9Disruption assayExtended sgRNAHeFSpCas9HeFm2SpCas9High fidelity nucleaseOff-targetSpCas9-HF1Truncated sgRNAeSpCas9sgRNA

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