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GOTI, a method to identify genome-wide off-target effects of genome editing in mouse embryos


Genome editing holds great potential for correcting pathogenic mutations. We developed a method called GOTI (genome-wide off-target analysis by two-cell embryo injection) to detect off-target mutations by editing one blastomere of two-cell mouse embryos using either CRISPR–Cas9 or base editors. GOTI directly compares edited and non-edited cells without the interference of genetic background and thus could detect potential off-target variants with high sensitivity. Notably, the GOTI method was designed to detect potential off-target variants of any genome editing tools by the combination of experimental and computational approaches, which is critical for accurate evaluation of the safety of genome editing tools. Here we provide a detailed protocol for GOTI, including mice mating, two-cell embryo injection, embryonic day 14.5 embryo digestion, fluorescence-activated cell sorting, whole-genome sequencing and data analysis. To enhance the utility of GOTI, we also include a computational workflow called GOTI-seq ( for the sequencing data analysis, which can generate the final genome-wide off-target variants from raw sequencing data directly. The protocol typically takes 20 d from the mice mating to sequencing and 7 d for sequencing data analysis.

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Fig. 1: Schematics of the GOTI method.
Fig. 2: Isolation of embryonic cells.
Fig. 3: Anticipated results from GOTI.

Data availability

The sequencing data were deposited in the National Center for Biotechnology Information’s Sequence Read Archive under project accession SRP119022 and

Code availability

The GOTI-seq pipeline is publicly available in GitHub at The code in this protocol has been peer reviewed.


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We thank the FACS facility in ION. This work was supported by the R&D Program of China (2018YFC2000100 and 2017YFC1001302 to H.Y. and 2017YFC0908405 to W.W.), the CAS Strategic Priority Research Program (XDB32060000), the National Natural Science Foundation of China (31871502 and 31522037), the Shanghai Municipal Science and Technology Major Project (2018SHZDZX05), the Shanghai City Committee of Science and Technology Project (18411953700, 18JC1410100) and a National Institutes of Health P01 Center grant (P01HG00020527 to L.M.S.).

Author information

Authors and Affiliations



E.Z. designed and performed experiments. Y.S., W.W., H.S. and L.Y. performed data analysis. T.Y. performed PCR analysis. W.Y. performed mouse embryo transfer. H.Y., Y.L. and L.M.S. supervised the project and designed experiments. E.Z., Y.S. and W.W. wrote the paper.

Corresponding authors

Correspondence to Lars M. Steinmetz, Yixue Li or Hui Yang.

Ethics declarations

Competing interests

L.M.S. has consulted for companies on CRISPR editing. The remaining authors declare no competing financial interests.

Additional information

Peer review information Nature Protocols thanks T. J. Cradick, Caixia Gao and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Zuo et al. Science 364, 289−292 (2019):

Zuo et al. bioRxiv (2020):

Integrated supplementary information

Supplementary Fig. 1 Primers for the generation of editing reagents.

a, The diagram of Cas9 primers. b, Primer diagram for sgRNA. c, The upstream primer sequence for sgRNA.

Supplementary Fig. 2 The three step sequential gating strategy for the isolation of mouse embryo cells.

a, Size fractionation on the basis of forward scatter Height (FSC-Height) and side scatter Height (SSC-Height). b, Elimination of cell debris or aggregates on the basis of SSC height and area. c, Selection of tdTomato+ Cells and tdTomato cells. The FSC/SSC gates of the starting cell population were set to include all cells. Then, doublet cells were excluded by SSC-H versus SSC-A. Positive and negative boundaries were defined by control progeny cells of non-edited blastomeres.

Supplementary Fig. 3 Quality control for in vitro transcribed RNA.

a–d, Preparation of in vitro transcription templates for SpCas9 (a), BE3 (b), Cre (c) and sgRNA (d). e, Amount and quality check for SpCas9, BE3, Cre and sgRNA transcripts. f, Failed in vitro transcription of spCas9 mRNA. For a successful in vitro transcription, a major band of interested transcript should appear like in (e), or it will be a failed reaction (f). In gel running results, smear could often be found around the major band of tailed transcripts, and size changes of transcripts might appear for RNA folding. The gene editing ability of these transcripts would not be changed as long as the major band could be clearly distinguished. DNA and RNA were run on 1% denaturing agarose stained with nucleic acid dye.

Supplementary Fig. 4 Zygote collection and processing diagram.

Isolate oviducts and place all the oviducts into one 200-μl drop of M2 medium in the 100-mm dishes (Step 19). Then, transfer one oviduct at a time into the second 200-μl drop of M2 medium. Tear the oviduct where it is most swollen using a 1-ml syringe attached to a 26-gauge needle, releasing the ZCCs. Repeat this step for every oviduct to get all the zygotes (Step 20). Add 200 μl of hyaluronidase to the ZCCs in the droplet, and then place the dish into a 37°C incubator for 3 min. Then, pipette the droplet up and down several times with a yellow tip until the cumulus cells are completely removed from the zygotes. Successively transfer the zygotes clockwise through five wash drops of M2 medium (Steps 21 and 22). Yellow circle, M2 medium drop. Red bar, oviduct. Green dot, ZCC.

Supplementary Fig. 5 Data quality of sequencing reads.

a, Sequencing reads with good quality. b, Unqualified sequencing reads need to be trimmed.

Supplementary Fig. 6 Venn diagrams of SNVs detected in embryos by WGS data using the indicated software tools.

The SNVs identified by all three algorithms—Mutect2, Strelka2 and Lofreq—are considered true SNVs. a, SNVs detected in Cas9-Tyr-C-treated embryo15. b, SNVs identified in BE3-Tyr-C-#1-treated embryo15.

Supplementary Fig. 7 Unsatisfactory results of FACS.

Left, FACS analysis with too few tdTomato+ cells. Right, FACS results with too many tdTomato+ cells.

Supplementary information

Supplementary Information

Supplementary Figs. 1–7.

Reporting Summary

Supplementary Video 1

Gene editing system components are introduced into one blastomere of two-cell mouse embryos via microinjection.

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Zuo, E., Sun, Y., Wei, W. et al. GOTI, a method to identify genome-wide off-target effects of genome editing in mouse embryos. Nat Protoc 15, 3009–3029 (2020).

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