Base-Edited Isogenic hPSC Line Generation (BIG-TREE)

Description:

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

Dr. Wang’s laboratory webpage  

 

Case ID:
M20-204L^
Published:
03-23-2021
Last Updated:
03-23-2021

For More Information, Contact