Abstract
SummaryCombined with CRISPR-Cas9 technology and single-stranded oligodeoxynucleotides (ssODNs), specific single-nucleotide alterations can be introduced into a targeted genomic locus in induced pluripotent stem cells (iPSCs); however, ssODN knockin frequency is low compared with deletion induction. Although several Cas9 transduction methods have been reported, the biochemical behavior of CRISPR-Cas9 nuclease in mammalian cells is yet to be explored. Here, we investigated intrinsic cellular factors that affect Cas9 cleavage activity in vitro. We found that intracellular RNA, but not DNA or protein fractions, inhibits Cas9 from binding to single guide RNA (sgRNA) and reduces the enzymatic activity. To prevent this, precomplexing Cas9 and sgRNA before delivery into cells can lead to higher genome editing activity compared with Cas9 overexpression approaches. By optimizing electroporation parameters of precomplexed ribonucleoprotein and ssODN, we achieved efficiencies of single-nucleotide correction as high as 70% and loxP insertion up to 40%. Finally, we could replace the HLA-C1 allele with the C2 allele to generate histocompatibility leukocyte antigen custom-edited iPSCs.
Highlights
Human induced pluripotent stem cells are important research tools for studying diseases and a promising cell source for regenerative medicine (Takahashi et al, 2007; Takahashi and Yamanaka, 2016)
We found that, ribonucleotide protein (RNP) transfection resulted in 13-fold lower levels of Cas9 protein compared with plasmid DNA (Figure 1A), the cleavage activity of RNP was 1.5-fold higher than that of plasmid DNA transfection by T7E1 analysis (Figure 1B)
With 13.3 mg single-stranded oligodeoxynucleotide (ssODN), we found that MaxCyte gave better knockin efficiency than 4D-Nucleofector in induced pluripotent stem cells (iPSCs) (1383D2) for both S691C (Figure S3E) and G694A (Figure S3F) substitution experiments
Summary
Human induced pluripotent stem cells (iPSCs) are important research tools for studying diseases and a promising cell source for regenerative medicine (Takahashi et al, 2007; Takahashi and Yamanaka, 2016). Because more than half of human pathogenic mutations are single-nucleotide polymorphisms (SNPs) (Rees and Liu, 2018), the introduction of an SNP into iPSCs is of high importance. Such genetic manipulation can be utilized for correcting a pathogenic mutation or introducing a desired mutation. Several studies have been previously reported to achieve successful ssODN-mediated HDR in iPSCs by utilizing cell-cycle regulators (Lin et al, 2014; Yang et al, 2016), chemical inhibitors to suppress NHEJ (Ma et al, 2018; Riesenberg and Maricic, 2018; Yu et al, 2015), sib selection (Miyaoka et al, 2014, 2016), optimization of transfection conditions (Li et al, 2016; Okamoto et al, 2019), chemical modification of ssODN molecules (Carlson-Stevermer et al, 2017; Savic et al, 2018), and a stably integrated inducible SpCas expression vector (Bertero et al, 2016; Chen et al, 2015; Dow et al, 2015; Ishida et al, 2018), since high levels of SpCas expression in target cells are thought to be correlated with high levels of genome editing activity in general
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