Highly efficient and specific genome editing in human cells with paired CRISPR-Cas9 nickase ribonucleoproteins

Abstract

CRISPR technology has opened up many diverse genome editing possibilities in human somatic cells, but has been limited in the therapeutic realm by both potential off-target effects and low genome modification efficiencies. Recent advancements to combat these limitations include delivering Cas9 nucleases directly to cells as highly purified ribonucleoproteins (RNPs) instead of the conventional plasmid DNA and RNA-based approaches. Here, we extend RNP-based delivery in cell culture to a less characterized CRISPR format which implements paired Cas9 nickases. The use of paired nickase Cas9 RNP system, combined with a GMP-compliant non-viral delivery technology, enables editing in human cells with high specificity and high efficiency, a development that opens up the technology for further exploration into a more therapeutic role.

Recent Advances in Genome-Editing Technology with CRISPR/Cas9 Variants and Stimuli-Responsive Targeting Approaches within Tumor Cells: A Future Perspective of Cancer Management

“…These variants create single-stranded breaks (SSBs) rather than double-strand breaks (DSBs). SpCas9 nickase includes a D10A point mutation that produces RuvC nuclease domain inactivation; thus, this form of nickase cleaves only the target DNA [ 68 ]. For the generation of DSB, two adjacent gRNAs are used with paired nickases.…”