The RNA-guided Cas9 nuclease from the microbial CRISPR (clustered regularly interspaced short palindromic repeats)-Cas system holds promise as a system that can be used for both gene knock out and activation ^1,2. Once bound to its target DNA, the active sgRNA-Cas9 complex can induce a double stranded break (DSB) via its RuvC and HNH domains. By mutating these nuclease domains, Cas9 can be made catalytically inactive^3,4 and repurposed for genetic perturbation beyond DNA editing. For example, this ‘dead’ Cas9 (dCas9) has been combined with protein domains that suppress or activate gene expression^5-9 or change the epigenetic state of a target locus¹⁰.

The simplest CRISPR-based orthogonal gene control system would function with only catalytically active wild type Cas9, in contrast to previous work that used distinct Cas9 proteins from different organism². Here we show that by reducing the length of the RNA targeting sequence to 14-15 nucleotides (nt), and by adding MS2 binding loops into the sgRNA backbone¹¹, sgRNAs can guide catalytically active Cas9 to activate transcription without inducing DSBs (Fig. 1a). After systematically studying how the design of these ‘dead sgRNAs’ (dRNAs) influences transcriptional activation and indel formation, and characterizing off-target transcriptional perturbations, we show that this system can simultaneously knock out and upregulate target genes in melanoma cells. These data demonstrate that sgRNAs can be engineered to exert transcriptional control using active Cas9, and that a single active Cas9 protein can be used for orthogonal gene control in mammalian cells.

Changes in sgRNA structure and mismatches between the sgRNA targeting sequence and DNA can prevent Cas9- mediated DNA cleavage^12-14. However, it is unknown whether these modified sgRNAs still allow binding of Cas9 to the DNA target. To test this, we designed sgRNAs with two structural characteristics. First, we added two aptamers that selectively bind dimerized MS2 bacteriophage coat proteins to the tetraloop and stem loop two of the sgRNA as previously described (sgRNA-MS2)¹¹. Second, we shortened the length of the sgRNA guide sequence from 20nt to 11nt. We reasoned that these changes could result in a dRNA that would still enable binding of Cas9 while preventing nuclease activity (Fig. 1a).

We transfected eighty sgRNA-MS2s targeting four DNA sequences within 200 bp of the transcriptional start site of human hemoglobin 1 (HBG1) together with active Cas9 and the MS2-P65-HSF1 (MPH) activation complex. The MPH complex has previously been reported to mediate efficient target upregulation by binding to MS2 loops in the sgRNA¹¹. We observed that guides from 20nt to 16nt resulted in indel formation, whereas shorter guides (11nt to 15nt) did not show detectable levels of indel formation in most cases (Fig. 1b). Notably, guides truncated to 11-15nt of complementarity to the target DNA were able to increase HBG1 mRNA expression by as much as 10,000 fold (Fig. 1c, Supplementary Fig. 1). We then investigated guides with mismatches on the 5’ end of the sgRNA analogous to our truncation experiments (Supplementary Fig. 2). In accordance with our results from truncated guides, we observed that guides with only 15bp complementarity to the target DNA were still able to mediate efficient activation in all four cases.

We proceeded to investigate the gene activation efficiency of 14 and 15nt dRNAs at three loci. In all cases the dRNAs, when co- transfected into HEK293FT cells with active Cas9 and the MPH complex, increased target mRNA expression of all three human genes (HBG1, Interleukin 1B (IL1B), and Zinc Finger Protease 42 (ZFP42)) without inducing significant indel formation (Fig. 1d). Notably, dRNA activation was comparable to the recently reported system using dCas9 in combination with a 20nt sgRNA-MS2¹¹. At all three loci 20nt sgRNAs cut target DNA and did not activate gene expression when combined with active Cas9. This was true for sgRNAs with and without the MS2 binding loops (Fig. 1d).

Biological studies utilizing activators will require specific target upregulation. Therefore, it is important to understand the specificity of Cas9 mediated gene activation. In addition, recent work has demonstrated that a single sgRNA can bind many sites in the genome, but the relationship between binding and transcriptional control is not clear¹⁵. Specificity may also change when the sgRNA is shortened to 15 bp. To assess the difference in specificity between 20nt sgRNA-MS2 and 15nt dRNAs we compared whole transcriptome mRNA levels in HEK293FT cells. Cells were co-transfected with dCas9, the MPH complex, and a 20nt activator sgRNA-MS2, or active Cas9, the MPH complex, and 15nt dRNA targeting the same sequence in the human HBG1/2 promoter. We previously determined that HBG1/2 upregulation induces limited downstream effects that could confound our analysis in HEK293FT cells. RNA-seq results showed that both the sgRNA/dCas9 and dRNA systems significantly activated HBG1/2 only, demonstrating that dRNAs can specifically upregulate target genes (Fig. 2a). We next performed off-target analysis on a second 15nt dRNA and 20nt sgRNA targeting the same HBG1/2 promoter. We found a significant number of perturbed transcripts for both the 15nt and 20nt guide RNAs (Fig. 2b)

We expanded this analysis by studying whole transcriptome analysis on ten additional sgRNAs targeting the proximal promoter of HBG1/2 (Supplementary Fig. 3). Overall, four out of twelve 20bp guides exhibited high specificity (<3 significant genome-wide off-targets), confirming activators can be specific. Notably, a previously published algorithm that predicts off-target indels did not correlate with the number of non-targeted transcripts that were altered by each guide (R = 0.12, p = 0.7)¹³.

Finally, we aimed to test whether dRNAs in combination with sgRNAs could mediate orthogonal gene control (activation and knockout) using only active Cas9. We previously used CRISPR-Cas9 loss-of-function (LOF)¹⁶ and gain-of-function (GOF)¹¹ screens to identify genetic modifiers that promote resistance of A375 melanoma cells to the BRAF inhibitor PLX-4720. We performed orthogonal gene regulation with hits selected from these screens. We first transduced and selected A375 cells with two lentiviral constructs encoding active Cas9 and the MPH complex, respectively (Fig. 2c). We then transduced these cells with lentiviral constructs encoding a dRNA targeting LPAR5 for activation and / or sgRNAs targeting MED12 or TADA2B for gene knockout. LPAR5 mRNA expression increased over 600-fold when cells were treated with dRNA targeting LPAR5, even when combined with sgRNAs targeting other genes. In all conditions, LPAR5 indels equal to 0.85% were detected (Fig. 2d). Indel rates of 0.6% and 0.05% at targeted loci were also measured after cells were treated with two additional dRNAs targeting EGFR and ITGA9, respectively (Supplementary Fig. 4). By contrast, the loci targeted by MED12 and TADA2B showed robust indel formation between 33-36% and 67.4-91.5%, respectively-even in the orthogonal conditions (Fig.2e).

dCas9 proteins have been engineered for diverse functions including gene suppression, gene activation, and epigenetic modification^5-10. Here we have demonstrated that guide RNAs can be engineered to bind target DNA and successfully recruit a transcriptional activation complex, without inducing measurable indel formation using an active Cas9 nuclease. These dRNAs can be designed to specifically and potently upregulate gene expression (Fig. 1, 2). We anticipate that dRNAs may also be designed to perform other genetic functions, for example, epigenetic modification or mRNA suppression.

dRNAs can be used to simplify orthogonal gene control experiments, as nuclease-mediated gene knockout and transcriptional activation can be achieved in the same cell population with one Cas9 protein (Fig. 3). By activating LPAR5, a gene previously described to promote drug resistance when upregulated¹¹, and knocking out known tumor suppressors¹⁶, we showed that orthogonal gene modifications can be performed in a biologically meaningful context (Fig. 3b-d). Streamlining orthogonal gene control is particularly important for in vivo experiments, since concurrently delivering two distinct Cas9 complexes to the same cell is challenging. Given the fact that mice have been engineered to constitutively and inducibly express Cas9^17,18, we anticipate that dRNAs can be utilized for in vivo orthogonal experiments, as the MPH activator complex combined with a dRNA and sgRNA fits within the 4.7kb packaging limit of AAV vectors.

Finally, our data align with previously reported work¹⁹ that show sgRNAs with seventeen or more nucleotides reliably and efficiently cut target DNA. It is notable that by reducing the guide length down to 15 nucleotides, we abrogate indel formation, but do not abrogate ‘functional binding’. Further evidence supporting this effect was recently published; active Cas9 fused to the transcriptional activator VPR and targeted to DNA by 14bp guide RNAs resulted in transcriptional activation²⁰. This may point to a more fundamental mechanism, whereby the interactions between the protein, sgRNA, and DNA dictate whether the Cas9 protein cuts the target DNA.