Structure and Engineering of Francisella novicida Cas9

Area of Science:

• Structural Biology
• Molecular Biology
• Genome Engineering

Background:

• The Cas9 enzyme, guided by RNA, performs double-stranded DNA cleavage for programmable genome editing.
• Cas9 activity is constrained by the requirement for a specific protospacer adjacent motif (PAM) sequence.
• Understanding Cas9-PAM interactions is crucial for expanding its genome editing applications.

Purpose of the Study:

• To determine the high-resolution crystal structures of Francisella novicida Cas9 (FnCas9) complexed with guide RNA and DNA targets.
• To compare FnCas9 structure with other Cas9 orthologs to identify conserved and divergent features.
• To engineer an FnCas9 variant with altered PAM specificity and demonstrate its utility in genome editing.

Main Methods:

• X-ray crystallography was used to obtain 1.7 Å resolution structures of FnCas9-guide RNA-DNA complexes.
• Structural comparisons were performed between FnCas9 and other known Cas9 orthologs.
• Site-directed mutagenesis was employed to create FnCas9 variants with modified PAM recognition.
• Microinjection of FnCas9-ribonucleoprotein complexes into mouse zygotes was used for in vivo genome editing.

Main Results:

• The crystal structures revealed conserved and divergent features of FnCas9 compared to other Cas9 orthologs.
• FnCas9 was found to recognize the 5'-NGG-3' PAM sequence.
• A variant FnCas9 was engineered to recognize the relaxed 5'-YG-3' PAM sequence.
• Microinjection of the engineered FnCas9 into mouse zygotes successfully edited endogenous sites with the 5'-YG-3' PAM.

Conclusions:

• Structural insights into FnCas9 provide a deeper understanding of CRISPR-Cas9 system diversity.
• Engineering FnCas9 for relaxed PAM recognition significantly expands the range of targetable genomic sites.
• The modified FnCas9 system holds promise for broader applications in genome editing, including in mammalian systems.