A protocol to generate SPH-OminiCMV-Ents mESCs

Detection of genetic information in the natural context is essential to understand the biological principles. However, the ability to sense the activity of endogenous genes is limited for conventional tools. Here, we developed a highly programmable sgRNA switch system (Ents) that enables detection of endogenous genes and lncRNAs, even with very low expression level. This protocol is related to the publication “Endogenous promoter-driven sgRNA for monitoring the expression of low-abundant genes and lncRNAs” in Nature Cell Biology.


Introduction
Manipulation of endogenous genetic information is crucial to understand the basic principles of biological systems. However, approaches to maniplulate endogenous information are limited, especially for the low-abundant genes and long non-coding RNAs (lncRNAs). Previous studies have shown that lncRNAs could play essential roles in various biological processes [1][2][3][4] . Nevertheless, functional characterization of a large population of lncRNAs remains challenging, mainly due to their low expression levels [5][6][7] .
Here, we developed an endogenous transcription-gated switch (Ents) by driving sgRNAs expression under an endogenous promoter. When Ents is paired with the highly-sensitive reporter, we could detect the activity of endogenous protein coding genes and lncRNAs faithfully in living cells, including genes with very low expression levels (< 0.001, relative to Gapdh, qPCR analysis). This system bypasses the procedure of identifying e cient sensor-actuator pairs, and offers virtually an unlimited number of highly effective triggers. We showed that this system could be applied to monitor the dynamic changes of genes with different expression levels in mouse ESCs (mESCs), providing a powerful platform to detect the information of various genetic elements in the natural context. In this protocol, we described detailed procedures for generating Ents system in mESCs. Seed mESCs on 6-well plate, using LIF-supplemented medium.
Replace with 2 ml LIF-supplemented medium 12 hours after transfection.

Day 8
Digest mESCs with 0.05% trypsin for 4 minutes at 37 °C, inactivate trypsin and centrifuge at 1000 rpm for 3 min, then seed mESCs to new 6-well plate.

Day 10
Digest mESCs with 0.05% trypsin and prepare for FACS into 96-well plates.
As previously described, Homology-Mediated End Joining (HMEJ) or homology recombination (HR) strategy (800 bp homology arm) was used to insert the sgRNA precursor, as previously described 8 .
Replace with 1 ml LIF-supplemented medium 12 hours after transfection.

Day 4
Digest mESCs with 0.05% trypsin and prepare for FACS, and sort positive cells, then seed GFP positive cells into 12-well plates.

Day 18
Sort single cells into 96-well plates by FACS, successful insertion of the sgRNA precursor is determined by PCR.

Day 22
Remove single colonies from 96-well plates to 24-well plates. Con rm sgRNA precursor positive colonies by PCR analysis.

Day 27
Measure the uorescent intensity of SPH-OminiCMV-Ents colonies by FACS, and take the uorescence images under a confocal microscope (Olympus FV3000).

Troubleshooting
Please use SaKKHCas9 to insert the SpCas9 sgRNA precursor Time Taken Around one and a half months

Anticipated Results
We could faithfully detect the activity of endogenous genes and lncRNAs.