Abstract
Target-specific genome editing using engineered nucleases has become widespread in various fields. Long gene knock-in and single-base substitutions can be performed by homologous recombination (HR), but the efficiency is usually very low. To improve the efficiency of knock-in with single-stranded oligo DNA nucleotides (ssODNs), we have investigated optimal design of ssODNs in terms of the blocking mutation, orientation, size, and length of homology arms to explore the optimal parameters of ssODN design using reporter systems for the detection of single-base substitutions. We have also investigated the difference in knock-in efficiency among the delivery forms and methods of Cas9 and sgRNA. The knock-in efficiencies for optimized ssODNs were much higher than those for ssODNs with no blocking mutation. We have also demonstrated that Cas9 protein/sgRNA ribonucleoprotein complexes (Cas9-RNPs) can dramatically reduce the re-cutting of the edited sites.
Highlights
CRISPR-Cas[9] genome editing holds promise in a wide variety of fields[1,2,3,4]
To evaluate the knock-in efficiency obtained with single-stranded oligo DNA nucleotides (ssODNs) using the CRISPR/Cas[9] system, we have established evaluation systems using ZsGreen[1] as a target gene for the simultaneous detection of accurate single-base substitutions and insertions and deletions (Indels) mutations in both 293T cells and human iPSCs (hiPSCs) using a flow cytometer
Using plasmids expressing Cas[9] and sgRNA, we have demonstrated that blocking mutations in ssODNs to prevent the re-cutting of the edited site can greatly improve the knock-in efficiency, and ssODNs with a mutation at PAM showed the highest knock-in efficiency using reporter systems
Summary
Cas9-RNPs can solve problems such as the random integration and insertion of plasmids into the genome at on-/ off-target sites and the severe cytotoxicity caused by the introduction of nucleic acids. In addition to those advantages, we have evaluated the feasibility of using Cas9-RNPs for single-base substitutions. For successful genome editing in induced pluripotent stem cells (iPSCs), in addition to improving the editing efficiency, increasing the single-cell cloning efficiency and maintaining iPSCs in an undifferentiated state throughout the whole process are essential[25,26,27,28]. ® culture system for hiPSCs, the Cellartis DEF-CSTM culture system in the single-cell cloning of genome-edited human iPSCs (hiPSCs) to obtain undifferentiated and correctly edited clones
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