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Editor’s note: Justin Smith and his colleagues at the Stanford Genome Technology Center and EMBL recently published “Quantitative CRISPR interference screens in yeast identify chemical-genetic interactions and new rules for guide RNA design” in Genome Biology. Justin talked with Benchling to explain how to design effective guides for CRISPRi by considering chromatin state and position of transcription start site. He also showed us how to perform functional, high-throughput CRISPRi screens in yeast effectively using an inducible single plasmid Justin and his colleagues created. Additional thanks to Raeka Aiyar, communications director at the Stanford Genome Technology Center, for writing help!
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As you probably already know, you can use an engineered guide RNA (gRNA) to guide the bacterial type II CRISPR (Clustered Regularly Interspaced Palindromic Repeats) associated Cas9 nuclease to specific DNA sequences for genome editing in a variety of organisms [1–4].
But beyond genome editing, Cas9 can also act as a programmable activator or repressor of gene expression, which The Atlantic recently highlighted as one of CRISPR’s “most exciting uses.” Just two point mutations yield a catalytically dead Cas9 (dCas9) [3] that serves as an effective programmable transcriptional repressor in bacteria [5]. With further modifications, dCas9 can function as a transcriptional activator or repressor in eukaryotes [6–10], including Saccharomyces cerevisiae [6, 7] (Figure 1).
Targeted gene repression using CRISPR is referred to as CRISPR interference (CRISPRi). Because many gRNAs can be synthesized on microarrays and the gRNAs themselves can serve as DNA barcodes, the system provides a powerful method to interrogate many different genes simultaneously. It is especially useful for essential genes that are lethal when deleted and thus cannot be interrogated with traditional deletion screens.
To repress transcription in eukaryotes, Gilbert et al. fused dCas9 to the human Mxi1 repressor (dCas9-Mxi1) [6], which was reported to interact with the histone deacetylase (Sin3 homolog in yeast) to repress transcription.
In our paper, we designed and constructed a plasmid for the expression of both dCas9-Mxi1 and gRNA under control a tetracycline inducible RNA polymerase III promoter: In the absence of anhydrotetracycline (ATc), the tet-repressor (TetR) binds the gRNA promoter and prevents PolIII from binding and transcribing the gRNA. This in turn prevents dCas9-Mxi1 from binding the target site. In the presence of ATc, TetR dissociates and gRNA is expressed, associates with dCas9-Mxi1 allowing it to bind its target locus, and repress gene expression (Figure 2).
To repress transcription in a rapid and regulatable way, we created a one plasmid system for expression of the dCas9-Mxi1 and the tetracycline repressor (TetR), as well as a tetracycline inducible gRNA locus containing the RPR1 promoter with a TetO site, a NotI site for cloning new gRNA sequences encoding target complementarity, and the constant part of the gRNA (Figure 3).
The system was designed to be compatible with Gibson Assembly using the protocol available on Benchling. If you are interested in using our system, you can order the plasmid from Addgene and the plasmid map is available on Benchling with all the primers we used.
To test how effective it is to induce gene repression in yeast in our plasmid system, we used qPCR to analyze the dynamics of gene expression following ATc induction and ATc removal. Gene expression is repressed within 2.5 hours following ATc addition with ~10-fold reduction (Figure 4).
Not all gRNAs work equally well, and this is particularly true for CRISPRi where gRNAs need to be targeted in such a way that dCas9 can repress transcription. We carried out a high-throughput CRISPRi screen to determine what factors influence gRNA efficacy – which hadn’t been systematically studied in yeast.
By screening hundreds of gRNAs tiling around the transcription start sites (TSS) of 20 genes, we were able to investigate the association of several features with gRNA efficacy. We assessed the effects of chromatin structure using ATAC-seq data, which provides a genome-wide map of chromatin accessibility [15]. Guides that target regions with low nucleosome occupancy and high chromatin accessibility were more effective. We also found that the best region to target gRNAs is between the TSS and 200 bp upstream of it (Figure 5).
Additionally, we tested the specificity of truncated gRNAs. Unlike nuclease-proficient Cas9 in human cells, the specificity of truncated gRNAs (18 nt of complementarity to the target) is not clearly superior to full-length gRNAs (20 nt of complementarity) with CRISPRi in yeast, as truncated gRNAs are generally less potent against both mismatched and perfectly matched targets and do not confer higher specificity.
Here is our workflow for parallel analysis of CRIPSRi-induced fitness defects in pooled cultures (Figure 6):
For a full explanation of how we determined these rules, please see our full manuscript in Genome Biology.
Don’t forget to check out our webtool that designs effective gRNAs in yeast based on these rules here.