Increasing CRISPR Efficiency and Measuring Its Specificity in HSPCs Using a Clinically Relevant System.

Genome editing of human cluster of differentiation 34 (CD34) hematopoietic stem and progenitor cells (HSPCs) holds great therapeutic potential. This study aimed to optimize on-target, genome editing using the CRISPR-Cas9 system in CD34 HSPCs and to create a clear workflow for precise identification of off-target effects. Modified synthetic guide RNAs (gRNAs), either 2-part gRNA or single-guide RNA (sgRNA), were delivered to CD34 HSPCs as part of ribonucleoprotein (RNP) complexes, targeting therapeutically relevant genes. The addition of an Alt-R electroporation enhancer (EE), a short, single-stranded oligodeoxynucleotide (ssODN), significantly increased editing efficiency in CD34 HSPCs. Notably, similar editing improvement was observed when excess gRNA over Cas9 protein was used, providing a DNA-free alternative suitable for therapeutic applications. Furthermore, we demonstrated that sgRNA may be preferable over 2-part gRNA in a locus-specific manner. Finally, we present a clear experimental framework suitable for the unbiased identification of bona fide off-target sites by Genome-Wide, Unbiased Identification of Double-Strand Breaks (DSBs) Enabled by Sequencing (GUIDE-seq), as well as subsequent editing quantification in CD34 HSPCs using rhAmpSeq. These findings may facilitate the implementation of genome editing in CD34 HSPCs for research and therapy and can be adapted for other hematopoietic cells.