A protocol to build microRNA-inducible CRISPR-Cas9 platform CURRENT

microRNAs (miRNAs) are small noncoding RNAs that play important regulatory roles in plants, animals and viruses. However, measuring miRNA activity in vivo remains a big challenge. In this protocol, using a miRNA-mediated sgRNA releasing strategy and dCas9-VPR to drive a transgene RFP expression, we create a miRNA sensor that can faithfully measure miRNA activity at cellular levels. When sgRNAs are designed to target endogenous locus, we show this system can be adapted to achieve cell type specific activation of endogenous genes. Furthermore, when dCas9 is fused with a transcriptional repressor or a base editor, we show this system can be used to repress the expression of endogenous genes or mutate specific DNA bases of chromosome upon induction by cell type-specific miRNAs. This step-by-step protocol is related to the publication “A microRNA-inducible CRISPR-Cas9 platform serves as microRNA sensors and cell type specific genome regulation tools” in Nature Cell Biology.

Introduction miRNAs are 21-24 nucleotide small RNAs regulating gene expression majorly at post-transcriptional level 1,2 . The partial complementarity between a miRNA and its target mRNA leads to the destabilization and/or translational repression of the target mRNA, while the full or near full complementarity between a miRNA and its target mRNA leads to the cleavage of the target mRNA at specific positions [3][4][5] . miRNAs play essential roles in a variety of physiologic and pathologic processes in plants and animals [6][7][8] . Many miRNAs are only expressed in specific tissues, cell types, and developmental or disease stages 9,10 . As a result, miRNA profiles have been successfully used to characterize the developmental lineage and differentiation status of human tumors, and in many cases are more accurate and informative than mRNA profiles 11 . A reporter for miRNA activity will be extremely useful for tracking differentiation status of stem cells and disease progression. However, due to the inhibitory nature of miRNAs on gene expression, a reporter that is activated by miRNAs has not been made possible. Furthermore, expansion the application of miRNAs rather than just as inhibitory tools remains to be challenging.
The type II CRISPR system has recently been repurposed to a programmable gene editing system in 3 plants, animals and microbes 12,13 . Guided by a programmable chimeric CRISPR single guide RNA (sgRNA), a Cas9 nuclease is recruited and induces a double-stranded break (DSB) at complementary genomic sequences. Furthermore, the nuclease-deficient Cas9 (dCas9) retains the ability to target genomic DNA without cleaving it 14 . When fused with different effector proteins, dCas9 system has been engineered as a platform to regulate transcription 15,16 , modify epigenetic status 17,18 and mutate DNA bases at specific genomic loci 19,20 . Traditionally, U6 or H1 promoters are used to driven sgRNAs expression while sgRNAs transcribed by Pol II RNA polymerase are inactive 21 , probably due to the 5' Cap and 3' polyA tail structures. Here, using a miRNA-mediated sgRNA releasing strategy from the inactive pre-sgRNA, we created a CRISPR-Cas9 platform that can be turned on by specific endogenous or exogenous miRNA/siRNAs. We show that this system can be adapted as miRNA sensors and cell type-specific genome regulation tools.
As illustrated in Figure 1a

Day1
Plate 50,000 mouse embryonic stem cells in 24-well plates, using standard growth medium.

Day2
Replace with 500 μl of growth medium.

Day8
Plate 500 cells on feeder for colony picking.

Day1
Plate 50,000 HEK293T cells per well in poly-D-lysine-coated 48-well plate. Add the solution to the cells carefully. 8 After 6 hours, replace medium with standard growth medium. Prepare A mix with 125 ng dCas9-VPR plus 125 ng pre-sgR Day4 48 hours after transfection, cells were harvested with Trizol for extracting total RNA for mRNA qRT-PCR \(mRNA Universa For endogenous gene repression, use dCas9-KRAB instead of dCas9-VPR.

Day1
Plate 50,000 Hela cells per well in 24-well plate.  Heat up the reaction to 95° C, using thermocycler for 5 minutes and let it cool down to room temperature.
Add 1 μl ApaI to the reaction, incubate 25° C for 3 hours.
Run digested product on 2% agarose gel under 120V for 30 minutes.