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Continuous directed evolution of a compact CjCas9 variant with broad PAM compatibility.

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CRISPR-Cas9 genome engineering advances with evoCjCas9, a variant of Campylobacter jejuni Cas9 (CjCas9). This enhanced CRISPR tool expands targeting range and improves in vivo gene editing for genetic diseases.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • CRISPR-Cas9 technology offers potential for genetic disease correction.
  • Limitations include restricted protospacer adjacent motif (PAM) recognition and large Cas9 size hindering in vivo delivery.

Purpose of the Study:

  • To broaden the PAM compatibility of Campylobacter jejuni Cas9 (CjCas9) using directed evolution.
  • To develop a smaller, more versatile Cas9 variant for enhanced genome engineering applications.

Main Methods:

  • Phage-assisted continuous directed evolution was employed to engineer CjCas9.
  • The evolved variant, evoCjCas9, was characterized for PAM specificity and nuclease activity.

Main Results:

  • The evoCjCas9 variant recognizes N4AH and N5HA PAM sequences, increasing genomic targeting frequency tenfold compared to wild-type CjCas9.
  • evoCjCas9 demonstrated enhanced nuclease activity and comparable editing rates to PAM-relaxed SpCas9 variants.
  • Small evoCjCas9 base editors facilitated efficient A-to-G and C-to-T transitions via single adeno-associated virus vectors.

Conclusions:

  • The engineered evoCjCas9 significantly expands CRISPR-Cas9 targeting scope and efficiency.
  • Its small size and broad PAM compatibility enable advanced applications like base and prime editing for genetic disease therapies.