An innovative approach using CRISPR-ribonucleoprotein packaged in virus-like particles to generate genetically...

Area of Science:

• Genetics
• Molecular Biology
• Biotechnology

Background:

• Genetically engineered mouse models (GEMMs) are essential tools for biological research and disease modeling.
• Current CRISPR gene editing techniques for GEMM creation face technical challenges and efficiency limitations.

Purpose of the Study:

• To establish a simplified and efficient CRISPR-VLP-induced targeted mutagenesis (CRISPR-VIM) strategy for generating GEMMs.
• To demonstrate the versatility of CRISPR-VIM for various genome editing applications in mice.

Main Methods:

• Co-culturing zygotes with virus-like particle (VLP)-delivered gene editing ribonucleoproteins (RNPs).
• Utilizing SpCas9, adenine base editor (ABE), and cytosine base editor (CBE) systems delivered via VLPs.
• Phenotypic characterization and germline transmission analysis of generated mouse models.

Main Results:

• Successfully generated Plin1 and Tyr knockout mice using VLP-delivered SpCas9 or ABE RNPs.
• Demonstrated successful C-to-T substitution and knock-in using VLP-delivered CBE and SpCas9 RNPs, respectively.
• Validated germline transmission of edited alleles.

Conclusions:

• The CRISPR-VIM strategy offers a simplified, non-damaging, and efficient approach to GEMM generation.
• This method accelerates the creation of mouse models without requiring specialized equipment or techniques.
• CRISPR-VIM has broad applicability across various research fields requiring precise genome editing in mice.