Sequential delivery of Cas9 or Cas12a RNPs and donor plasmids improved virus-free knock-in editing of clinical-grade iPSCs.
Evidence
This iPSC gene-editing platform experiment optimized RNP and donor-plasmid delivery, achieved full-length transgene knock-ins above 30%, and generated iPSC clones lacking HLA class I with an inducible caspase-9 safety switch.
Caveat
It is a workflow and cell-line engineering study rather than a therapy test, and performance beyond the tested GMP iPSC lines and edits is not established.
Simplified
Human induced pluripotent stem cells (iPSCs) are gaining momentum as a powerful starting material in cell therapy. To fully harness their potential, CRISPR technology permits endogenous gene modifications as well as the introduction of advanced features, to increase the immune compatibility of the cells or insert suicide genes for enhancing therapeutic safety, for instance. However, genetic manipulation of iPSCs, in particular the generation of knock-in lines, remains relatively inefficient. Conventional mitigation strategies, such as enriching for positive cells using antibiotic selection or complex instrumentation, may, however, cause conflicts with good manufacturing practice () requirements. To address this challenge, we have systematically optimized a basic gene editing procedure using both Cas9 and Cas12a-based ribonucleoprotein (RNP) complexes. Based on the sequential delivery of RNPs and donor plasmids as a critical hallmark, this virus-free approach permits knock-ins of full-length transgenes at above 30% efficiency, while readily identifying positive clones through random screening at small scale. We exemplify these advances by creating and characterizing homozygous iPSC lines depleted of HLA class I and carrying an inducible caspase-9 suicide gene. Isolated clones from independent GMP iPSC lines retained genomic integrity, differentiation capability, and functionality of the safety switch in the differentiated state. This improved methodology will form a flexible platform for custom gene editing universally applicable both in basic iPSC research and therapy.
Key numbers
30%
Efficiency
Efficiency of knock-ins in using the optimized workflow.
5 of 39 wells
Negative Clones
Proportion of wells containing negative clones after gene editing.
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