A. High efficiency on-target editing in bulk HSPC populations

B. Successful gene editing in clonal isolates

C. Characterization of off-target InDels

D. Characterization of off-target single nucleotide variants

Genome-wide sequencing analysis of CRISPR-Cas9 activity in human HSPCs.
Editing efficiency was determined in A) bulk (pre-clonal) HSPC and B) cellular clones using a T7E1 assay. On-target InDel formation in bulk HSPCs was 67% for CXCR4 (left) and 58% AAVS1 (right). Gene editing efficiencies were high, given the accepted limits of sensitivity of the T7E1 assay. Clonal isolates selected for WGS analysis are indicated by a color-coded dot. No EP=non-electroporated cells; EP Only=Cells electroporated without CRISPR RNP; Cas9 Only=Cells electroporated without gRNA. WGS revealed a total of C) 4474 off-target InDels and D) 15,855 off-target single nucleotide variants (SNVs) distributed among all the samples. The majority of InDels and SNVs appeared within intergenic or non-protein coding regions within the genome. Electroporated cells and untreated cells displayed similar distributions of both InDels and SNVs regardless of the presence of Cas9 RNPs.
Summary
- The high efficiency and low toxicity of MaxCyte® electroporation ensures highly targeted gene disruption frequencies with negligible off-target effects that bolster therapeutic efficacy.
- Gene editing using MaxCyte non-viral engineering enables rapid development of next-generation adoptive cell therapies for treatment of a wide variety of diseases.
- MaxCyte electroporation technology efficiently delivers a diversity of payloads including mRNA, sgRNA, and RNPs to difficult-to-engineer primary cells commonly used for adoptive cell therapies including hematopoietic stem cells and T cells.
- MaxCyte electroporation enables high levels of gene editing including:
- Gene knockout/disruption
- Gene knockin
- Single nucleotide gene mutation correction
- Development and optimization of MaxCyte electroporation for new cell types and payloads is a rapid, straightforward process
References:
Smith R, Chen Y, Seifuddin F, et al. Genome-Wide Analysis of Off-Target CRISPR-Cas9 Activity in Single-Cell-Derived Human Hema- topoietic Stem and Progenitor Cell Clones. Genes. 2020;11(12):1501. Published 2020 Mar 13. doi :10.3390/genes112150