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. 2026 Apr 13;33(5):756–767. doi: 10.1038/s41594-026-01788-6

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© The Author(s) 2026

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 1 — a. Cas12f-MG119-1. b. Cas12f-MG119-2. c. Cas12f-MG119-3. d. Al3Cas12f. PAM sequences (pink Ns in schematics) were confirmed by NGS and represented as SeqLogos. SeqLogos were generated from separate cleavage reactions treated with the Klenow fragment or the Mung Bean nuclease to confirm the target strand (TS) cut site or the non-target strand (NTS) cut site, respectively as shown in Supplementary Fig. 2. As expected, the PAM sequence was the same with either treatment. Histograms show the number of NGS reads (counts) mapped to the cleavage site on each strand. Carrots over sequences in the cut sites schematics denote the cut sites. Cas12f-MG119-3 cut site on the NTS was less than a 1000 reads, therefore the SeqLogo and histogram are not shown, however the assumed cut sites are indicated with gray carrots. The cut site on the TS was determined to be 22, 23 and 11, 13 on the NTS based on the number of reads mapped at these locations. In the cut site schematics, the U40 spacer sequence is shown in blue font, and the target DNA sequence is shown in orange font.