CRISPR-Cas12a for Highly Efficient and Marker-Free Targeted Integration in Human Pluripotent Stem Cells

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Cellular and Genetic Therapies

Background:

• The CRISPR-Cas12a system is a powerful tool for genome editing, recognized for its staggered DNA double-strand break mechanism targeting AT-rich PAM sequences.
• The development of engineered Cas12a variants, such as Cas12a Ultra, has expanded its applicability in diverse cell types, including primary human cells.

Purpose of the Study:

• To evaluate the efficiency of CRISPR-Cas12a Ultra in gene disruption and knock-in applications within human T cells, hematopoietic stem and progenitor cells (HSPCs), and induced pluripotent stem cells (iPSCs).
• To demonstrate the potential of CRISPR-Cas12a Ultra for precise gene integration, specifically targeting the AAVS1 safe harbor site and the CSF2RA gene in human iPSCs.

Main Methods:

• Utilized CRISPR-Cas12a Ultra ribonucleoprotein complexes for gene editing experiments in human cell lines and primary cells.
• Assessed gene disruption frequencies at multiple target sites using quantitative methods.
• Performed targeted knock-in experiments, integrating a GFP marker and a CSF2RA super-exon into specific genomic locations in human iPSCs without selection.

Main Results:

• Achieved allelic gene disruption frequencies exceeding 90% in human T cells, HSPCs, and iPSCs.
• Demonstrated efficient targeted integration of a GFP gene into the AAVS1 site and a CSF2RA super-exon into CSF2RA in up to 90% of alleles in human iPSCs, without the need for selection.
• Confirmed bi-allelic integration in over 50% of screened iPSC clones, maintaining pluripotency and genomic integrity.

Conclusions:

• CRISPR-Cas12a Ultra is a highly efficient genome editing platform for both gene disruption and targeted knock-ins in human iPSCs.
• The combination of CRISPR-Cas12a Ultra with adeno-associated virus vectors offers a robust strategy for precise gene integration in stem cell applications.
• This technology holds significant potential for advancing therapeutic genome engineering in human cells.