CRISPR/Cas9-Based Protocol for Precise Genome Editing in Induced Pluripotent Stem Cells

Area of Science:

• Molecular Biology
• Stem Cell Biology
• Genetic Engineering

Background:

• CRISPR/Cas9 technology offers powerful genome editing capabilities.
• Editing human induced pluripotent stem cells (iPSCs) with CRISPR faces challenges in cell survival and efficiency.
• P53 activation during CRISPR editing can induce apoptosis, hindering success.

Purpose of the Study:

• To develop a protocol to enhance cell survival and editing efficiency in human iPSCs using CRISPR.
• To mitigate the apoptotic response triggered by CRISPR editing.
• To streamline the creation of isogenic cell lines.

Main Methods:

• Combining p53 inhibition with pro-survival small molecules during CRISPR editing.
• Utilizing CRISPR/Cas9 technology for targeted genome modification in human iPSCs.
• Assessing homologous recombination rates and cell survival post-editing.

Main Results:

• A novel protocol combining p53 inhibition and pro-survival molecules significantly enhances cell survival.
• Achieved a homologous recombination rate exceeding 90% in human iPSCs.
• Reduced the overall time for iPSC genome editing to as little as 8 weeks.

Conclusions:

• The combination of p53 inhibition and pro-survival molecules is an effective strategy for improving CRISPR editing outcomes in iPSCs.
• This optimized protocol accelerates the generation of genetically modified iPSCs.
• The findings facilitate the efficient creation of isogenic cell lines for research and therapeutic applications.