If you’re using CRISPR to make a genome edit, how do you know if your CRISPR experiment was successful in your organism or cell type? You can use DNA sequencing or other molecular cloning techniques to determine CRISPR/sgRNA efficiency of an experiment and confirm the correct edit was made to the genome without any off target effects. However, these methods can be labor intensive and quite time consuming.
As Cas9/gRNA activity is crucial for successful CRISPR editing experiments, there was a real need in the scientific community for efficient real-time Cas9 activity assays. Recently scientists have created CRISPR fluorescent reporter assays to quantify targeted Cas9 cleavage, and repair through homology directed repair (HDR) and non-homologous end joining (NHEJ). In these reporters, editing typically results in a change in the fluorescent readout.
In this blog we will focus on two fluorescent CRISPR reporter methods, SRIRACCHA and GEMCherry2. We will cover single base-pair editing CRISPR reporters in the near future.
SRIRACCHA (stable, but reversible, integrated reporter for assaying CRISPR/Cas-stimulated HDR activity)
The Madison lab created SRIRACCHA in 2017 to enrich and identify desired CRISPR mutants (Wen et al., 2018). SRIRACCHA uses the Piggybac transposon to stably integrate a reporter gene that contains a puromycin resistance gene followed by the target site (the same sequence you’d like to mutate in the genome) and an out-of-frame H2B (histone)-GFP reporter into the genome of mammalian cells.
Figure 1: The SRIRACCHA reporter construct. Image from Wen et al., 2017.
Once you’ve introduced the reporter into the cells, you’re ready to edit. These cells are then transfected with several different constructs for the reporter to work and to make the desired genome edit:
- A donor plasmid that contains a puromycin resistance gene linked to an in-frame H2B-GFP. This visual read out allows for easy screening of different gRNAs by simply changing the gRNA used when transfecting your cells.
- A plasmid that contains Cas9. The Madison lab also created an inducible SRIRACCHA system (iSRIRACCHA) where Cas9 expression is activated using 4-OH-T and doxycycline.
- A gRNA that targets your desired gene (whose sequence is also found in the reporter gene).
- A control RFP expression plasmid.
With this set up, all successfully transfected cells will express RFP while cells containing the reporter gene that was cleaved by Cas9 and repaired via HDR using the supplied donor plasmid will express GFP. If there’s GFP expression, presumably the endogenous target site will be cut and repaired by NHEJ as well. The ratio of RFP to GFP expressing cells provides a real-time read out of Cas9 activity.
By using the RFP:GFP ratio, edited cells can be sorted from unedited cells. In an experiment that targeted the gene NFE2L2, the Madison lab found that H2B-GFP expressing cells exhibited a 2-4 fold increase in the number of endogenous indel edits when compared to unsorted cells (Wen et al., 2018). Thus SRIRACCHA enables scientists to both quantify Cas9 activity and sgRNA efficiency as well as enrich for mutants through the presence of a fluorescent reporter. Lastly, once a mutant is identified, the PB transposase can easily and seamlessly remove the integrated GFP reporter leaving you with just your desired genomic edit.
GEMCherry2, an improved monomeric fluorescent protein for assessing CRISPR/Cas9 activity
Guide RNA structure and the site in the genome you are trying to target play a large role in Cas9 activity and efficiency (Sander and Joung, 2014). Thus it is essential to identify the best sgRNA to ensure success in your CRISPR experiments. But how? There have been several systems to do this but most are labor-intensive, require multiple fluorescent proteins, and aren’t that efficient. To overcome these issues the Denham lab developed a modified monomeric red fluorescent protein (RFP), named GEMCherry2, that can rapidly and efficiently evaluate Cas9 cutting sites and activity (Knudsen et al., 2018).
To create GEmCherry2, the Denham lab first incorporated an out-of-frame CRISPR genomic target region into the N-terminus of mCherry. To prevent alternative start site initiation, the lab removed the native methionine start site. This first iteration was named GEmCherry1, due to the “genomic fragment inserted into mCherry”. However this one mutated methionine was not enough to prevent alternative initiation, so they mutated the methionines at position 14 and 21 to create GEmCherry2. The Denham lab made several other small changes creating 2 other versions but these did not surpass GEmCherry2 in background fluorescence and in-frame fluorescence.
|Figure 2: The GEmCherry reporter. Image from Højland Knudsen et al., 2018.|
So how does GEmCherry2 work? The premise is that the genomic region inserted into the N-terminus of mCherry can be targeted by a sgRNA guided Cas9 causing a DSB break. This DSB is repaired by NHEJ which depending on the nucleotides inserted or deleted, can result in an in-frame shift of mCherry and thus expression. As a proof of concept the Denham lab compared editing at three different genomic sites in the AAVS1 locus using GEmCherry2. In this experiment they saw a clear difference in mCherry expression using FACs sorting, confirming yet again that sgRNA structure and genomic recognition sites play a large role in CRISPR-Cas9 efficiency. They continued to use GEmCherry2 to identify the most efficient sgRNAs to target a specific locus to create a knock-out in human embryonic stem cells.
Overall SRIRACHA and GEmCherry2 reporter systems provide efficient, rapid, and real-time read-outs of Cas9/gRNA efficiency. So if you are looking to identify the most effective gRNA and enrich cells for your CRISPR mutant of interest, give these methods a try.
Højland Knudsen C, Ásgrímsdóttir ES, Rahimi K, Gill KP, Frandsen S, Hvolbøl Buchholdt S, Chen M, Kjems J, Febbraro F, Denham M (2018) A Modified Monomeric Red Fluorescent Protein Reporter for Assessing CRISPR Activity. Frontiers in Cell and Developmental Biology 6: . https://doi.org/10.3389/fcell.2018.00054
Sander JD, Joung JK (2014) CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology 32:347–355 . https://doi.org/10.1038/nbt.2842
Wen Y, Liao G, Pritchard T, Zhao T-T, Connelly JP, Pruett-Miller SM, Blanc V, Davidson NO, Madison BB (2017) A stable but reversible integrated surrogate reporter for assaying CRISPR/Cas9-stimulated homology-directed repair. Journal of Biological Chemistry 292:6148–6162 . https://doi.org/10.1074/jbc.m117.777722
Additional resources on the Addgene blog
- Browse all of our CRISPR blog posts
- Find more about fluorescent proteins
- Get a refresher on CRISPR with our CRISPR 101 blog posts
Resources on Addgene.org
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