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Engineered CRISPR-Cas9 nuclease with expanded targeting space.

Hiroshi Nishimasu1, Xi Shi2,3, Soh Ishiguro4,5,6

  • 1Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. nisimasu@bs.s.u-tokyo.ac.jp nureki@bs.s.u-tokyo.ac.jp.

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Scientists engineered a Cas9 enzyme variant (SpCas9-NG) to recognize relaxed NG protospacer adjacent motifs (PAMs). This expands genome editing capabilities by targeting previously inaccessible genomic loci in human cells.

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Area of Science:

  • Molecular Biology
  • Genomics
  • Biochemistry

Background:

  • The Cas9 enzyme is a key tool in genome editing, enabling targeted DNA cleavage.
  • The commonly used Streptococcus pyogenes Cas9 (SpCas9) enzyme is limited by its requirement for a specific NGG protospacer adjacent motif (PAM).
  • This NGG PAM specificity restricts the range of genomic sites that can be targeted for editing.

Purpose of the Study:

  • To engineer a SpCas9 variant with relaxed PAM recognition capabilities.
  • To overcome the limitations imposed by the strict NGG PAM requirement of the wild-type SpCas9.
  • To expand the scope of targetable genomic loci for CRISPR-based genome editing.

Main Methods:

  • Rational engineering of the SpCas9 enzyme to create a variant (SpCas9-NG) with altered PAM specificity.
  • X-ray crystallography to determine the structural basis of NG PAM recognition by SpCas9-NG.
  • Functional assays in human cells to assess SpCas9-NG's ability to induce indels at NG PAM sites.
  • Fusion of SpCas9-NG with activation-induced cytidine deaminase (AID) to test for base editing capabilities at NG PAM sites.

Main Results:

  • Successfully engineered SpCas9-NG variant capable of recognizing relaxed NG PAMs.
  • Crystal structure revealed that SpCas9-NG accommodates NG PAMs through altered base-specific interactions.
  • SpCas9-NG demonstrated efficacy in inducing insertions/deletions (indels) at endogenous NG PAM sites in human cells.
  • SpCas9-NG fused with AID facilitated targeted C-to-T conversions at NG PAM sites in human cells.

Conclusions:

  • SpCas9-NG represents a significant advancement in Cas9-based genome editing technology.
  • The engineered SpCas9-NG broadens the targeting range, enabling editing at previously inaccessible genomic loci.
  • SpCas9-NG fusion with AID offers a versatile tool for precise base editing at NG PAM sites, enhancing genome engineering applications.