Abstract
Highly efficient gene knockout (KO) editing of CRISPR–Cas9 has been achieved in iPSCs, whereas homology-directed repair (HDR)-mediated precise gene knock-in (KI) and high-level expression are still bottlenecks for the clinical applications of iPSCs. Here, we developed a novel editing strategy that targets introns. By targeting the intron before the stop codon, this approach tolerates reading frameshift mutations caused by nonhomologous end-joining (NHEJ)-mediated indels, thereby maintaining gene integrity without damaging the non-HDR-edited allele. Furthermore, to increase the flexibility and screen for the best intron-targeting sgRNA, we designed an HDR donor with an artificial intron in place of the endogenous intron. The presence of artificial introns, particularly an intron that carries an enhancer element, significantly increased the reporter expression levels in iPSCs compared to the intron-deleted control. In addition, a combination of the small molecules M3814 and trichostatin A (TSA) significantly improves HDR efficiency by inhibiting NHEJ. These results should find applications in gene therapy and basic research, such as creating reporter cell lines.
Highlights
Induced pluripotent stem cells provide an ideal source for cell replacement therapy and regenerative medicine due to their unlimited self-renewal and multidirectional differentiation ability [1]
The homology-directed repair (HDR) donor vectors contained E2A-mNeonGreen flanked by homology arms (HAs) omitting introns (Fig. 1a, top)
Pearson linear regression analysis showed that HDR editings were proportional to indel efficiencies (R2 = 0.35, P = 0.0004) (Fig. 2a), suggesting that the single-guide RNA (sgRNA) targeting ability largely dictates HDR efficiency
Summary
Induced pluripotent stem cells (iPSCs) provide an ideal source for cell replacement therapy and regenerative medicine due to their unlimited self-renewal and multidirectional differentiation ability [1]. The realization of the full therapeutic potential of human iPSCs requires further development of approaches to generate gene-modified or disease gene-corrected cells. The clustered regularly interspaced short palindromic repeats (CRISPR)–Cas system has become a valuable tool for gene editing, from manipulating human cell genomes to creating gene-modified animal models. The CRISPR–Cas genome editing system, developed from the adaptive immune system of bacteria and archaea, consists of a Cas nuclease and a single-guide RNA (sgRNA) [5]. SpCas nuclease introduces double-stranded DNA breaks (DSBs) 3 bp upstream of the NGG protospacer adjacent motif (PAM) under the guidance of sgRNA. Since broken DNA is a dangerous signal for cells and causes severe cytotoxicity [7, 8], the DNA
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