Human pluripotent stem cells (hPSCs) are a vital tool not only in research and disease modeling but also in developmental biology and regenerative medicine because of their ability to self-renew and differentiate. Current genome editing tactics in hPSCs require the incorporation of deleterious double-stranded DNA breaks followed by inefficient homology-directed repair (HDR). These methods can result in DNA insertion or deletion issues, apoptosis, oncogenic mutations and other negative outcomes.
Researchers at Arizona State University have developed a novel method using CRISPR tools to co-target genomic loci and an episomal reporter to enable single-nucleotide genomic changes in hPSCs without HDR. This method, called base-edited isogenic hPSC line generation using a transient reporter for editing enrichment (BIG-TREE), enables the rapid generation of clonal isogenic hPSC lines. It produces single-nucleotide editing efficiencies greater than 80% across multiple hPSC lines and allows for the efficient generation of loss-of-function hPSC lines and efficient multiplex editing of hPSCs at multiple independent loci.
BIG-TREE advances the implementation of base-editing technologies in hPSCs for use in developmental biology, disease modeling, drug screening, cell-based therapies and more.
Potential Applications
• Isogenic cell line engineering
• Generating knockout cell lines
• Developmental biology
• Disease modeling
• Drug screening
• Regenerative medicine
Benefits and Advantages
• Greater than 80% efficiencies across multiple hPSC lines
• Allows for the precise and efficient base editing of hPSCs
• Efficient generation of loss-of-function hPSC lines
• Efficient editing of multiple loci and across several independent hPSC lines
• Bulk enrichment of base-edited cell populations including hPSCs
• Fast and efficient generation of clonal isogenic hPSC lines with homozygous and heterozygous single base pair edits
• Rapid engineering of isogenic hPSC lines
o Establishes in vitro models to assess pathogenic risk/disease mechanisms
• Allows for biallelic/multiplexed targeting without the need for sequential retargeting
• Compatible with off the shelf chemical transfection reagents
o Doesn’t require cloning of complex viral constructs/use of specialized cell transfection systems
• Can be used in conjunction with other base editing variants that have altered PAM specificities and editing windows
For more information about this opportunity, please see
Brookhouseer et al – Stem Cell Reports – 2020
For more information about the inventor(s) and their research, please see
Dr. Brafman’s departmental webpage