Generation of genetically modified mice using SpCas9-NG engineered nuclease

Area of Science:

• * Molecular Biology
• * Genetics
• * Bioengineering

Background:

• * CRISPR/Cas9 genome editing is highly efficient for generating genetically modified mice.
• * Conventional CRISPR/Cas9 is limited to targeting loci with specific protospacer-adjacent motif (PAM) sequences (e.g., 5'-NGG).
• * Expanding the PAM recognition of Cas9 is crucial for broader genome editing applications.

Purpose of the Study:

• * To investigate the efficacy of engineered Streptococcus pyogenes Cas9 (SpCas9-NG) in mouse zygotes.
• * To determine if SpCas9-NG can target loci with alternative PAM sequences beyond the conventional NGG.
• * To assess the potential of SpCas9-NG for generating both knockout and knock-in mouse models.

Main Methods:

• * Utilized CRISPR/Cas9-mediated genome editing in mouse zygotes.
• * Employed engineered SpCas9-NG with altered PAM recognition.
• * Generated knockout and knock-in mouse models to evaluate SpCas9-NG functionality.

Main Results:

• * SpCas9-NG successfully recognized and targeted 5'-NGA, 5'-NGC, and 5'-NGT sequences as PAMs in mouse zygotes.
• * Genome editing with SpCas9-NG enabled the efficient generation of knockout mice at these expanded PAM sites.
• * SpCas9-NG facilitated the creation of knock-in mice at loci not targetable by conventional SpCas9.

Conclusions:

• * SpCas9-NG significantly broadens the targeting range for CRISPR/Cas9 genome editing in mouse zygotes.
• * This engineered nuclease is a valuable tool for generating diverse knockout and knock-in mouse models.
• * SpCas9-NG expands the utility of genome editing for creating genetically modified mice at NGN-PAM loci.