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

• Main Authors: Jenny Shapiro, Ortal Iancu, Ashley M. Jacobi, Matthew S. McNeill, Rolf Turk, Garrett R. Rettig, Ido Amit, Adi Tovin-Recht, Zohar Yakhini, Mark A. Behlke, Ayal Hendel
• Format: Article
• Language: English
• Published: Elsevier 2020-06-01
• Series: Molecular Therapy: Methods & Clinical Development
• Subjects: genome editing; CRISPR-Cas9; CD34+ hematopoietic stem and progenitor cells; chemically modified guide RNAs; off-target sites
• Online Access: http://www.sciencedirect.com/science/article/pii/S2329050120300875

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, ex vivo 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.