摘要：CRISPR/Cas9 efficiently generates gene knock-out via nonhomologous end joining (NHEJ), but the efficiency of precise homology-directed repair (HDR) is substantially lower, especially in the hard-to-transfect human stem cells and primary cells. Herein we report a tube electroporation method that can effectively transfect human stem cells and primary cells with minimal cytotoxicity. When applied to genome editing using CRISPR/Cas9 along with single stranded DNA oligonucleotide (ssODN) template in human induced pluripotent stem cells (iPSCs), up to 42.1% HDR rate was achieved, drastically higher than many reported before. We demonstrated that the high HDR efficiency can be utilized to increase the gene ablation rate in cells relevant to clinical applications, by knocking-out β2-microglobulin (B2M) in primary human mesenchymal stem cells (MSCs, 37.3% to 80.2%), and programmed death-1 (PD-1) in primary human T cells (42.6% to 58.6%). Given the generality and efficiency, we expect that the method will have immediate impacts in cell research as well as immuno- and transplantation therapies.
