Details

Autor(en) / Beteiligte

• Wei, Nan
• Shang, Lu
• Liu, Jing
• Wang, Mi
• Liu, Yingchun
• Zhu, Chuangang
• Fei, Chenzhong
• Zhang, Lifang
• Yang, Fayu
• Gu, Feng

Titel

Engineered Staphylococcus auricularis Cas9 with high‐fidelity

Ist Teil von

• The FASEB journal, 2023-08, Vol.37 (8), p.e23060-n/a

Ort / Verlag

United States

Erscheinungsjahr

2023

Link zum Volltext

Quelle

MEDLINE

Beschreibungen/Notizen

• CRISPR‐Cas9 is a versatile gene editing tool with a broad application of basic research and clinical therapeutics. However, the potential impact caused by off‐target effects remains a critical bottleneck. The small Cas9 ortholog from Staphylococcus auricularis (SauriCas9) was identified, which recognizes a 5′‐NNGG‐3′ protospacer adjacent motif (PAM), exhibiting high activity for genome editing. Recently, we also reported enhanced‐fidelity Staphylococcus aureus Cas9 (efSaCas9), which harbors a single mutation N260D. Protein sequence alignment revealed that SauriCas9 has 62.4% sequence identity with SaCas9. Because SauriCas9 is more flexible in recognizing the target sequence with PAM of 5′‐NNGG‐3′ than SaCas9 of 5′‐NNGRRT‐3′ PAM, we sought to test whether key mutation(N260D) or adjacent residue mutation in efSaCas9 can be appliable to SauriCas9. With this concept, two engineered SauriCas9 variants (SauriCas9‐HF1, harboring the N269D mutation; SauriCas9‐HF2, harboring the D270N mutation) dramatically improved targeting specificity by targeted deep sequencing and GUIDE‐seq. At certain sites, reduced off‐target effects (approximately 61.6‐ and 111.9‐fold improvements) of SauriCas9‐HF2 compared with wild‐type SauriCas9 were observed. Overall, two identified SauriCas9 variants (SauriCas9‐HF1 and SauriCas9‐HF2) expand the utility of the CRISPR toolkit for research and therapeutic applications. Based on the key mutation of SaCas9 variant, we successfully generated two high‐fidelity variants of SauriCas9 (SauriCas9‐HF1 and SauriCas9‐HF2), which expand the utility of the CRISPR toolkit for research and therapeutic applications.