Efficient ssODN-Mediated Targeting by Avoiding Cellular Inhibitory RNAs through Precomplexed CRISPR-Cas9/sgRNA Ribonucleoprotein

Abstract

Summary Combined 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.

Optimized electroporation of CRISPR-Cas9/gRNA ribonucleoprotein complex for selection-free homologous recombination in human pluripotent stem cells

“…Ribonucleoprotein (RNP) form of delivery has several advantages over the DNA/RNA form ( Gee et al., 2020 ; Kim et al., 2014 ; Paquet et al., 2016 ; Richardson et al., 2016 ; Zuris et al., 2015 ), since the RNP functions immediately, degrades quickly, and is relatively smaller than plasmid DNA or mRNA. Furthermore, the pre-formed Cas9/gRNA RNP complex can evade the inhibition by cellular mRNA ( Kagita et al., 2021 ).…”

“… Okita et al. (2011) From Dr. Okita, RRID:CVCL_DP92 Oligonucleotides T7-DMDsgRNA1 forward primer: GAAA TTAATACGACTCACTATAgggtatcttacag gaactccGTTTTAGAGCTAGAAATA GCAAG This Paper N/A T7-DMD-in55- g3-IVT forward primer: GAAA TTAATACGACTCACTATAggactttatagata tctcccaGTTTTAGAGCTAGAAATA GCAAG This Paper N/A T7-DYSFgRNA2-IVT forward primer: GAAATT AATACGACTCACTATAggtactacacacact gacggGTTTTAGAGCTAGAAAT AGCAAG This Paper N/A T7-sgRNA reverse primer: AAAGCACCGA CTCGGTGCCACTTTTTCAAGTTGATAAC GGACT AGCCTTATTTTAACTTGCTATTT CTAGCTCTAAAAC This Paper N/A DMD1+loxPssODN (134-mer): AAGAC ATGGGGCTTCATTTTTGTTTTGCCTTT TTGGTATCTTACAGGAACATAACTTC GTATAGCATACATTATACGAAGTTAT TCCAGGATGGCATTGGGCAGCGGC AAACTGTTGTCAGAACATTGAATGCA This Paper N/A DMD-in55- g3+loxP-ssODN (134-mer): TCA TTTGGAGGTAATTTGTTTGGAACAGTAT CAGACTTTATAGATATCTCATAACTTCGT ATAGCATACATTATACGAAGTTATCCATG GCTTGTGATAGAATATAAGGGCAATGC AAATGTAGAGTTTTTTGC Kagita et al. (2021) N/A DMDexon55(45-55)check_dir5 primer: CCTCGGGTACACTGAAAGTTATGTG This Paper N/A DMDexon45(45-55)check_rev5 primer: CACCACAGGCTTTAACTTCTGCCG This Paper N/A Software and algorithms CRISPRdirect Naito et al.…”

“…With the RNP and ssODN electroporation condition, extra bands with expected sizes (229 bp and 67 bp) were observed, suggesting the loxP sequence is inserted at the target site with around 24% of efficiency. This figure is adapted from Kagita et al. (2021) .…”

“…Here, we describe the optimized protocol to introduce a single nucleotide change or a loxP site insertion in feeder-free, xeno-free iPSCs by utilizing MaxCyte and 4D-Nucleofector electroporators. For complete details on the use and execution of this protocol, please refer to Kagita et al. (2021) and Xu et al.…”

“…For complete details on the use and execution of this protocol, please refer to Kagita et al. (2021) and Xu et al.…”