Genome-Wide Analysis of Off-Target CRISPR/Cas9 Activity in Single-Cell-Derived Human Hematopoietic Stem and Progenitor Cell Clones

• Published in: Genes Vol. 11; no. 12; p. 1501
• Main Authors: Smith, Richard H., Chen, Yun-Ching, Seifuddin, Fayaz, Hupalo, Daniel, Alba, Camille, Reger, Robert, Tian, Xin, Araki, Daisuke, Dalgard, Clifton L., Childs, Richard W., Pirooznia, Mehdi, Larochelle, Andre
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI 13.12.2020
• Subjects: bioinformatics; CRISPR-Cas systems; genes; genetic heterogeneity; genome-wide association study; genomics; humans; loci; ribonucleoproteins; statistical analysis; stem cells; CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9); off-target activity; whole genome sequencing (WGS)
• ISSN: 2073-4425; 2073-4425
• DOI: 10.3390/genes11121501

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)-mediated genome editing holds remarkable promise for the treatment of human genetic diseases. However, the possibility of off-target Cas9 activity remains a concern. To address this issue using clinically relevant target cells, we electroporated Cas9 ribonucleoprotein (RNP) complexes (independently targeted to two different genomic loci, the CXCR4 locus on chromosome 2 and the AAVS1 locus on chromosome 19) into human mobilized peripheral blood-derived hematopoietic stem and progenitor cells (HSPCs) and assessed the acquisition of somatic mutations in an unbiased, genome-wide manner via whole genome sequencing (WGS) of single-cell-derived HSPC clones. Bioinformatic analysis identified >20,000 total somatic variants (indels, single nucleotide variants, and structural variants) distributed among Cas9-treated and non-Cas9-treated control HSPC clones. Statistical analysis revealed no significant difference in the number of novel non-targeted indels among the samples. Moreover, data analysis showed no evidence of Cas9-mediated indel formation at 623 predicted off-target sites. The median number of novel single nucleotide variants was slightly elevated in Cas9 RNP-recipient sample groups compared to baseline, but did not reach statistical significance. Structural variants were rare and demonstrated no clear causal connection to Cas9-mediated gene editing procedures. We find that the collective somatic mutational burden observed within Cas9 RNP-edited human HSPC clones is indistinguishable from naturally occurring levels of background genetic heterogeneity.