Multiplex Genome Editing of Human Pluripotent Stem Cells Using Cpf1

Area of Science:

• * Molecular Biology
• * Genetics
• * Stem Cell Biology

Background:

• * Targeted genome editing in human pluripotent stem cells (hPSCs) is crucial for research and therapeutic applications.
• * Site-specific endonucleases, like Cpf1 (CRISPR from Prevotella and Francisella), are key tools for precise DNA modification.
• * Existing methods often require complex delivery systems for efficient editing.

Purpose of the Study:

• * To develop and validate a streamlined single-vector system for Cpf1-mediated genome editing in hPSCs.
• * To demonstrate efficient multiplex genome editing at multiple loci simultaneously.
• * To assess the precision and safety of the system, including off-target mutation analysis.

Main Methods:

• * A single-vector system was designed to co-deliver Cpf1 endonuclease and CRISPR RNA (crRNA).
• * The system utilized a U6 promoter-driven guide RNA array for targeting multiple genomic loci, including AAVS1 and MAFB.
• * Homologous recombination was employed for precise knockin editing, and indel formation for knockout editing.

Main Results:

• * Efficient multiplex genome editing was achieved at the AAVS1 (knockin) and MAFB (knockout) loci in hPSCs within a single experiment.
• * Edited hPSCs maintained pluripotency markers and demonstrated differentiation potential into neurons and functional β-cells (INS reporter cell line).
• * Rigorous screening identified no detectable off-target mutations in sequences homologous to the crRNA, indicating high specificity.

Conclusions:

• * The single-vector Cpf1 system offers an efficient and flexible approach for precise genome editing in hPSCs.
• * This method facilitates simultaneous multi-locus editing and subsequent cellular differentiation, advancing stem cell research.
• * The system's minimal off-target effects enhance its utility for both basic research and potential therapeutic applications.