A rapid and robust protocol for generating loss-of-function alleles in pluripotent stem cells

Area of Science:

• Stem cell biology
• Molecular genetics
• Gene editing technologies

Background:

• Generating loss-of-function alleles in human pluripotent stem cells (hPSCs) is crucial for understanding gene function and validating research tools.
• Current methods for identifying true loss-of-function alleles in hPSCs are often inefficient, hindering research progress.

Purpose of the Study:

• To present BOLT (barcoded oligos for loss-of-function targeting), a streamlined protocol designed to simplify and accelerate the screening process for loss-of-function mutations in hPSCs.
• To facilitate the rapid and accurate validation of gene knockouts in hPSCs.

Main Methods:

• The BOLT protocol involves designing gene editing tools, performing nucleofection, and clonal density plating.
• Key steps include bridging PCR, isolating single-cell clones, and employing Sanger barcode detection for efficient screening.

Main Results:

• The BOLT protocol significantly simplifies the screening process for identifying hPSC clones with true loss-of-function alleles.
• This method enables rapid validation of induced loss-of-function mutations, improving experimental efficiency.

Conclusions:

• BOLT offers a robust and efficient solution for generating and validating loss-of-function alleles in hPSCs.
• This protocol accelerates genetic studies in hPSCs, aiding in gene function discovery and reagent validation.