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Continuous evolution of SpCas9 variants compatible with non-G PAMs
Shannon M Miller1,2,3, Tina Wang1,2,3, Peyton B Randolph1,2,3
1Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA, USA.
Nature Biotechnology
|February 12, 2020
Summary
Scientists evolved new Streptococcus pyogenes Cas9 (SpCas9) variants. These variants expand gene editing capabilities by targeting a wider range of DNA sequences, including previously inaccessible ones for sickle cell anemia treatment.
Area of Science:
- Molecular Biology
- Biotechnology
- Genetics
Background:
- The CRISPR-Cas9 system from Streptococcus pyogenes (SpCas9) is a powerful gene editing tool.
- Its utility is limited by the requirement for specific protospacer-adjacent motif (PAM) sequences, primarily those containing guanine (G) bases.
Purpose of the Study:
- To evolve new SpCas9 variants with expanded PAM targeting capabilities.
- To overcome the limitations of existing SpCas9 systems for broader genomic applications.
Main Methods:
- Utilized phage-assisted non-continuous evolution (PANGE) to evolve SpCas9 variants.
- Employed three novel phage-assisted continuous evolution (PACE) strategies for DNA binding and secondary selection for DNA cleavage.
- Characterized variant targeting in HEK293T cells using a comprehensive library of 11,776 integrated protospacer-sgRNA pairs covering all NNNN PAMs.
Main Results:
- Successfully evolved three new SpCas9 variants that collectively recognize NRNH PAMs (R=A/G, H=A/C/T).
- Demonstrated indel formation and base editing in human cells with the evolved variants.
- Achieved therapeutic A•T-to-G•C base editing of a sickle cell anemia mutation using a previously inaccessible CACC PAM.
Conclusions:
- The evolved SpCas9 variants significantly broaden the targeting scope of Cas9-based gene editing.
- These variants, alongside existing ones, enable targeting of most NR PAM sequences, reducing inaccessible genomic sites.
- Opens new avenues for precise gene editing in research and therapeutic applications, including genetic diseases like sickle cell anemia.
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