From Design to Single-Cell Cloning: A Complete RNP-Based CRISPR-Cas9 Protocol for Precision Gene Correction in Human iPSCs.
TL;DR
A systematic, comprehensive and efficient workflow combining generation, genotyping and expansion of high-quality monoclonal iPSC lines following CRISPR-Cas9 genome editing, which provides a robust and high-throughput framework for the reliable production of monoclonal genome-edited iPSCs, thereby advancing translational research and the development of iPSC-based regenerative therapies and disease models.
Abstract
Precise genome editing of induced pluripotent stem cells (iPSCs) using clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR-Cas9) has opened unprecedented avenues for advancements in regenerative medicine and disease modelling. However, the establishment of isogenic single-cell-derived iPSC populations, particularly upon CRISPR-Cas9 gene editing modifications, is one of the major challenges still associated with these advanced methodologies relying on low-rate editing events and requiring defined clonogenicity. In response, we have developed a systematic, comprehensive and efficient workflow combining generation, genotyping and expansion of high-quality monoclonal iPSC lines following CRISPR-Cas9 genome editing. In particular, the protocol incorporates optimized single-cell cloning procedures for two commercially available dispensing platforms, one based on microfluidic imaging and the other on impedance technology, together with rapid droplet digital PCR (ddPCR)-based screening of non-homologous end joining (NHEJ) and homology-directed repair (HDR) outcomes. By combining gentle single-cell handling with advanced genotyping methodologies, the protocol enables efficient early assessment of editing outcomes before commitment to labour-intensive clonal derivation, thereby accelerating project timelines, minimising cell stress and loss, preserving genetic fidelity and supporting scalability. Coupled with precisely defined culture conditions tailored for post-seeding recovery, these approaches aim to improve iPSC viability and clonal outgrowth, achieving at least 60% in 96-well plate format within 10 days. Importantly, the protocol goes beyond step-by-step experimental instructions by providing comprehensive design strategies, decision-making criteria, and practical advice for avoiding and addressing the common pitfalls and unintended consequences of these advanced methods. Collectively, this integrated end-to-end approach provides a robust and high-throughput framework for the reliable production of monoclonal genome-edited iPSCs, thereby advancing translational research and the development of iPSC-based regenerative therapies and disease models.
