✍🏼 Erdene Baljinnyam, Laura Grisanti, Shalini Tattari, Chiara Pedicone, Bhavana Shewale, Nicole Dubois, Alison M. Goate, William G. Kerr, Samuele G. Marro

🏠 Institute for Regenerative Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA

📑 STAR Protocols (2025)

Abstract
The generation of loss-of-function alleles in human pluripotent stem cells (hPSCs) is used to interrogate gene function and validate reagents; however, identifying clones harboring true loss-of-function alleles remains inefficient. To address this, we present BOLT (barcoded oligos for loss-of-function targeting), a streamlined protocol that simplifies the screening process, facilitating rapid validation of loss-of-function mutations. We describe steps for designing editing tools, nucleofection, and clonal density plating. We then detail procedures for bridging PCR, isolating clones derived from a single hPSC, single-clone screening, and Sanger barcode detection.

How the WOLF is used in this study
In this protocol, the WOLF cell sorter was used after genome editing to isolate and enrich single pluripotent stem cells for clonal expansion, ensuring the derivation of true loss-of-function clones. Specifically, following CRISPR-Cas9 targeting and cell recovery, edited hPSCs were gently sorted as single cells into individual wells using the WOLF sorter to enable monoclonal outgrowth in a controlled, low-stress environment. This sorting step was essential to separate single, viable edited cells from unedited or heterogeneous populations, improving the efficiency of clonal screening and validation of loss-of-function alleles through downstream genotyping and Sanger sequencing.