Analysis of SpCas9 on- and off-target effects in high efficiency multiplex editing in Arabidopsis
- 1CEA Cadarache
Abstract
RNA-guided nucleases (RGNs), such as Cas9 from Streptococcus pyogenes (SpCas9), are widely used for plant genome editing. Previous surveys for off-targeting, the modification of unintended targets with similarity to the intended target, indicate high specificity of SpCas9 in plant cells. However, off-targeting has not been assessed for efficiency-optimized editing systems combined with extensive multiplexing, which might increase the likelihood of cleavage at unintended sites. We therefore analyzed Arabidopsis thaliana lines that had been extensively mutagenized using zCas9i and up to 29 gRNAs addressing >45 target sites over several rounds of editing. Genomes were sequenced by short- and long-read technologies, and genome-wide variants were catalogued. Our pipeline for variant calling reliably detected RGN-induced mutations at on-targets. When excluding these on-target modifications, variants were detected in edited lines at frequencies similar to those previously reported for spontaneous mutations. In further analyses, we did not find any evidence for an origin of these variants from RGN activity. Our data are thus consistent with high specificity of SpCas9. In contrast, we detected genomic reorganization events upon editing at two complex loci, RPP1 and RPP7, encompassing multiple homologous genes, and also identified an allele by WGS that had escaped detection by amplicon sequencing. We conclude that, while off-targets may efficiently be avoided by selection of specific gRNAs, on-target modifications may be more extensive than intended, especially at complex loci and/or during multiplexing.
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BibTeX
@misc{stuttmann2026analysis,
title = {{Analysis of SpCas9 on- and off-target effects in high efficiency multiplex editing in Arabidopsis}},
author = {J. Stuttmann and J. Keilwagen and P. Ortet and F.-S. Lim and T. Schmutzer and C. Llauro and A. Mbodj and M. Mirouze and F. Hartung and R. Wilhelm},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.28.754964},
url = {https://www.biorxiv.org/content/10.64898/2026.09.28.754964v1},
note = {CC BY-NC},
}
RIS
TY - UNPB
TI - Analysis of SpCas9 on- and off-target effects in high efficiency multiplex editing in Arabidopsis
AU - J. Stuttmann
AU - J. Keilwagen
AU - P. Ortet
AU - F.-S. Lim
AU - T. Schmutzer
AU - C. Llauro
AU - A. Mbodj
AU - M. Mirouze
AU - F. Hartung
AU - R. Wilhelm
PY - 2026
DA - 2026/09/28
PB - bioRxiv
DO - 10.64898/2026.09.28.754964
UR - https://www.biorxiv.org/content/10.64898/2026.09.28.754964v1
AB - RNA-guided nucleases (RGNs), such as Cas9 from Streptococcus pyogenes (SpCas9), are widely used for plant genome editing. Previous surveys for off-targeting, the modification of unintended targets with similarity to the intended target, indicate high specificity of SpCas9 in plant cells. However, off-targeting has not been assessed for efficiency-optimized editing systems combined with extensive multiplexing, which might increase the likelihood of cleavage at unintended sites. We therefore analyzed Arabidopsis thaliana lines that had been extensively mutagenized using zCas9i and up to 29 gRNAs addressing >45 target sites over several rounds of editing. Genomes were sequenced by short- and long-read technologies, and genome-wide variants were catalogued. Our pipeline for variant calling reliably detected RGN-induced mutations at on-targets. When excluding these on-target modifications, variants were detected in edited lines at frequencies similar to those previously reported for spontaneous mutations. In further analyses, we did not find any evidence for an origin of these variants from RGN activity. Our data are thus consistent with high specificity of SpCas9. In contrast, we detected genomic reorganization events upon editing at two complex loci, RPP1 and RPP7, encompassing multiple homologous genes, and also identified an allele by WGS that had escaped detection by amplicon sequencing. We conclude that, while off-targets may efficiently be avoided by selection of specific gRNAs, on-target modifications may be more extensive than intended, especially at complex loci and/or during multiplexing.
ER -