Combinatorial CRISPR/Cas12a excises targeted loci in the mammalian genome at very low efficiency
- 1Department of Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
Abstract
The CRISPR/Cas12a system has emerged as a highly useful tool for genomic editing due to its ability to process multiple guide RNAs (gRNAs) from a single promoter. In this study, we explored the potential of combinatorial CRISPR/Cas12a to excise targeted loci in the mammalian genome by inducing double-strand breaks (DSBs) upstream and downstream of targeted exons. We designed a library of gRNA pairs targeting introns flanking 54 exons of 18 essential genes and 17 exons of 10 nonessential genes, aiming to assess the efficiency of Cas12 in excising targeted loci. While single gRNAs directly targeting essential exons produced expected fitness defects in cells, pairs of guides targeting flanking intronic regions resulted in only trivial viability effects, suggesting that excision rarely occurred. Further analysis showed no meaningful correlation between gRNA efficiency, orientation, GC content, or cut site geometry and the success of locus excision. These findings indicate that, under the conditions tested, the Cas12a system does not yield locus excision at the efficiency required for sensitive negative selection assays.
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BibTeX
@misc{anvar2026combinatorial,
title = {{Combinatorial CRISPR/Cas12a excises targeted loci in the mammalian genome at very low efficiency}},
author = {N. Esmaeili Anvar and Z. M. Szegletes and R. J. Steger and A. V. MGee and S. Wang and J. G. Doench and T. Hart},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.21.753273},
url = {https://www.biorxiv.org/content/10.64898/2026.09.21.753273v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Combinatorial CRISPR/Cas12a excises targeted loci in the mammalian genome at very low efficiency
AU - N. Esmaeili Anvar
AU - Z. M. Szegletes
AU - R. J. Steger
AU - A. V. MGee
AU - S. Wang
AU - J. G. Doench
AU - T. Hart
PY - 2026
DA - 2026/09/21
PB - bioRxiv
DO - 10.64898/2026.09.21.753273
UR - https://www.biorxiv.org/content/10.64898/2026.09.21.753273v1
AB - The CRISPR/Cas12a system has emerged as a highly useful tool for genomic editing due to its ability to process multiple guide RNAs (gRNAs) from a single promoter. In this study, we explored the potential of combinatorial CRISPR/Cas12a to excise targeted loci in the mammalian genome by inducing double-strand breaks (DSBs) upstream and downstream of targeted exons. We designed a library of gRNA pairs targeting introns flanking 54 exons of 18 essential genes and 17 exons of 10 nonessential genes, aiming to assess the efficiency of Cas12 in excising targeted loci. While single gRNAs directly targeting essential exons produced expected fitness defects in cells, pairs of guides targeting flanking intronic regions resulted in only trivial viability effects, suggesting that excision rarely occurred. Further analysis showed no meaningful correlation between gRNA efficiency, orientation, GC content, or cut site geometry and the success of locus excision. These findings indicate that, under the conditions tested, the Cas12a system does not yield locus excision at the efficiency required for sensitive negative selection assays.
ER -