Most genome editing analyses to date are based on quantifying small insertions and deletions. Here, we show that CRISPR-Cas9 genome editing can induce large gene modifications, such as deletions, insertions, and complex local rearrangements in different primary cells and cell lines. We analyzed large deletion events in hematopoietic stem and progenitor cells (HSPCs) using different methods, including clonal genotyping, droplet digital polymerase chain reaction, single-molecule real-time sequencing with unique molecular identifier, and long-amplicon sequencing assay. Our results show that large deletions of up to several thousand bases occur with high frequencies at the Cas9 on-target cut sites on the HBB (11.7 to 35.4%), HBG (14.3%), and BCL11A (13.2%) genes in HSPCs and the PD-1 (15.2%) gene in T cells. Our findings have important implications to advancing genome editing technologies for treating human diseases, because unintended large gene modifications may persist, thus altering the biological functions and reducing the available therapeutic alleles.
Manipulation of cells for applications such as biomanufacturing and cell-based therapeutics involves introducing biomolecular cargoes into cells. However, successful delivery is a function of multiple experimental factors requiring several rounds of optimization. Here, we present a high-throughput multiwell-format localized electroporation device (LEPD) assisted by deep learning image analysis that enables quick optimization of experimental factors for efficient delivery. We showcase the versatility of the LEPD platform by successfully delivering biomolecules into different types of adherent and suspension cells. We also demonstrate multicargo delivery with tight dosage distribution and precise ratiometric control. Furthermore, we used the platform to achieve functional gene knockdown in human induced pluripotent stem cells and used the deep learning framework to analyze protein expression along with changes in cell morphology. Overall, we present a workflow that enables combinatorial experiments and rapid analysis for the optimization of intracellular delivery protocols required for genetic manipulation.
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