Rapid Generation of Human Genetic Loss-of-Function iPSC Lines by Simultaneous Reprogramming and Gene Editing

Area of Science:

• Stem cell biology
• Gene editing technologies
• Human disease modeling

Background:

• Studying gene function and loss-of-function (LOF) mutations in human induced pluripotent stem cells (iPSCs) is crucial for understanding genetic disorders.
• Existing methods for engineering iPSCs can be time-consuming and less efficient for creating disease models.

Purpose of the Study:

• To develop and validate a streamlined method for simultaneous iPSC reprogramming and CRISPR/Cas9-mediated gene editing.
• To enable the rapid and efficient generation of human cell models for loss-of-function genetic disorders.

Main Methods:

• Developed a novel technique combining iPSC reprogramming with CRISPR/Cas9 gene editing to introduce insertions/deletions (indels).
• Applied the method to target multiple genes, including those associated with epileptic encephalopathy and metabolic disorders.
• Generated heterozygous and homozygous loss-of-function iPSC lines with passage-matched isogenic controls.

Main Results:

• Achieved a high and consistent rate of successful gene editing across different target genes.
• Demonstrated greater efficiency and consistency compared to CRISPR/Cas9 editing in established iPSC lines.
• Successfully generated isogenic control lines alongside the mutagenized lines in a single step.

Conclusions:

• The developed method offers a rapid, efficient, and reproducible approach for creating loss-of-function human disease cell models.
• This technique facilitates the study of genetic diseases, even without access to patient-derived tissues.
• Provides ideal control lines for robust research into gene function and disease mechanisms.