On- and off-target effects of paired CRISPR-Cas nickase in primary human cells

• Published in: Molecular therapy Vol. 32; no. 5; pp. 1298 - 1310
• Main Authors: Klermund, Julia, Rhiel, Manuel, Kocher, Thomas, Chmielewski, Kay Ole, Bischof, Johannes, Andrieux, Geoffroy, el Gaz, Melina, Hainzl, Stefan, Boerries, Melanie, Cornu, Tatjana I., Koller, Ulrich, Cathomen, Toni
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.05.2024
• Subjects: CAST-seq; chromosomal aberrations; CRISPR-Cas; double nickase; epidermolysis bullosa; gene editing; off target; paired nickase; structural variants; translocations; structural variants; double nickase; epidermolysis bullosa; paired nickase; chromosomal aberrations; CRISPR-Cas; off target; translocations; CAST-seq; gene editing
• ISSN: 1525-0016; 1525-0024
• DOI: 10.1016/j.ymthe.2024.03.006

Undesired on- and off-target effects of CRISPR-Cas nucleases remain a challenge in genome editing. While the use of Cas9 nickases has been shown to minimize off-target mutagenesis, their use in therapeutic genome editing has been hampered by a lack of efficacy. To overcome this limitation, we and others have developed double-nickase-based strategies to generate staggered DNA double-strand breaks to mediate gene disruption or gene correction with high efficiency. However, the impact of paired single-strand nicks on genome integrity has remained largely unexplored. Here, we developed a novel CAST-seq pipeline, dual CAST, to characterize chromosomal aberrations induced by paired CRISPR-Cas9 nickases at three different loci in primary keratinocytes derived from patients with epidermolysis bullosa. While targeting COL7A1, COL17A1, or LAMA3 with Cas9 nucleases caused previously undescribed chromosomal rearrangements, no chromosomal translocations were detected following paired-nickase editing. While the double-nicking strategy induced large deletions/inversions within a 10 kb region surrounding the target sites at all three loci, similar to the nucleases, the chromosomal on-target aberrations were qualitatively different and included a high proportion of insertions. Taken together, our data indicate that double-nickase approaches combine efficient editing with greatly reduced off-target effects but still leave substantial chromosomal aberrations at on-target sites. [Display omitted] Genome editing is still challenged by off-target effects. While CRISPR-Cas9 double-nickase-based strategies are efficient, it has not been investigated whether they leave on-target aberrations. Cathomen and colleagues introduce a novel CAST-seq pipeline to show that dual-nickase approaches reduce off-target effects but generate significant chromosomal abnormalities at their target sites.