Programmable mammalian translational modulators by CRISPR-associated proteins

1.

Flow cytometry data analysis

Flow cytometry data sets were analyzed using FlowJo version 10.5.3

(BD Biosciences) and Excel (Microsoft). Gates were generated using

mock samples. Data from debris were eliminated when preparing

forward- versus side-scatter dot plots (FSC-A versus SSC-A). Then,

events on the chart edges in the dot plots of the EGFP intensity versus

the iRFP670 intensity were removed. In the histogram where iRFP670intensity is displayed on the X-axis, the iRFP670-positive (referencepositive) gate was defined (Supplementary Fig. 1C). In the following

analysis, the median of reporter/reference of each cell was calculated

from the reference positive population using FlowJo.

Relative reporter expression was defined using the following

formulas:

Normalized intensity ðNIÞ = 1000 ×

median of the ratioðreporter intensity=reference intensityÞ of each cell

2.

3.

4.

5.

6.

7.

ð8Þ

8.

Relative intensity ðRIÞ = ðNI of trigger + Þ=ðNI of triggerÞ

ð9Þ

Relative reporter expression = ðRIÞ=ðRI of }No gRNA} sampleÞ

ð10Þ

9.

10.

11.

In Supplementary Fig. 2, all RI values were normalized by the value

of the [Trigger -] [No gRNA] sample.

In Fig. 7D, fold activation was defined as RI value normalized by

the value of the [-,-,-] sample.

The fold change in Supplementary Fig. 17C was defined as RI in ON

switch and the reciprocal of RI in OFF switch.

In Fig. 6D, the net fold-change was calculated by dividing the NI in

ON state ([1,1]) by the averaged value of NI in OFF states ([0,0], [0,1],

and [1,0]). Vector proximity angle is the angle between two

4-dimensional vectors. One is a truth table vector that has ideal output

(= 0 or 1) for each state ([0,0], [1,0], [0,1], and [1,1]). The other is a

vector that carries the observed output levels (=NI) of each state ([0,0],

[1,0], [0,1], and [1,1]). Thus, θ ranges from 0˚ (best) to 90˚ (worst).

Nature Communications | (2023)14:2243

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Acknowledgements

We thank Dr. Peter Karagiannis, Dr. Kelvin K. Hui (Kyoto University), and

Dr. Zoher Gueroui (École Normale Supérieure) for critical reading of the

manuscript; Yusuke Shiba, Ryo Hirayama, and Shintaro Oe (Kyoto University) for helping with the experiments; and Miho Nishimura, Yuko

Kono, Hiromi Takemoto, and Shodai Komatsu (Kyoto University) for their

administrative support. We also thank Dr. Hideyuki Nakanishi (Tokyo

Medical and Dental University) for the intein information, Dr. Yoshihiko

Fujita (Kyoto University) for establishing the imaging quantification

method, Dr. Akitsu Hotta (Kyoto University) for providing the AsCas12a

ORF, and Shigetoshi Kameda (Kyoto University) for providing some IVT

templates. This work was supported by JSPS KAKENHI Grant Number JP

19K16110 (to S.K.), 19J21199 (to H.O.), 20K15777 (to M.H.), 15H05722, and

20H05626 (to H.S.), and the iPS Cell Research Fund.

https://doi.org/10.1038/s41467-023-37540-7

record listed on the patents. H.S. own shares of aceRNA Technologies

Ltd., and has outside director of aceRNA Technologies Ltd. The other

authors declare no competing interests.

Additional information

Supplementary information The online version contains

supplementary material available at

https://doi.org/10.1038/s41467-023-37540-7.

Correspondence and requests for materials should be addressed to

Shunsuke Kawasaki or Hirohide Saito.

Peer review information Nature Communications thanks Nozomu

Yachie, Chong Zhang and the other, anonymous, reviewer(s) for their

contribution to the peer review of this work. Peer reviewer reports are

available.

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Competing interests

Open Access This article is licensed under a Creative Commons

Attribution 4.0 International License, which permits use, sharing,

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long as you give appropriate credit to the original author(s) and the

source, provide a link to the Creative Commons license, and indicate if

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licenses/by/4.0/.

Kyoto University has filed a patent application regarding the CARTRIDGE

method (JP2020044905). S.K., H.O., M.H., and H.S. are the inventors of

© The Author(s) 2023

Author contributions

S.K. and H.S. managed the project. S.K. conceived the idea. M.H. found

the translational repression ability of SpCas9. S.K., H.O., M.H., and H.S.

designed the project. S.K., H.O., M.H., and T.K. performed the experiments and analyzed the data. S.S. performed bioinformatic analysis,

including calculation of MFE and the orthogonality verification. S.L. and

K.W. established the Dox-inducible dCas9 cell line. H.O. mainly prepared the figures under the supervision of S.K.. S.K., H.O., M.H., and H.S.

wrote the manuscript. S.K., H.O., and M.H. contributed equally to

this work.

Nature Communications | (2023)14:2243

17

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