IntroductionThe development of programmable Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas)-based transcriptional activation (CRISPRa) tools is of high interest for research and clinical applications1. In these approaches, transcriptional activators are recruited to specific sites in the genome, by fusion to endonuclease-inactivated Cas proteins (“dCas”, most commonly dCas92) or through aptamers in the associated single guide RNA (sgRNA)3,4,5,6,7. The appeal of CRISPRa over traditional cDNA expression approaches lies in its simplicity of sgRNA design, scalability, ability to multiplex, and ability to overexpress relevant gene isoforms and large transcripts from their endogenous loci. CRISPRa can furthermore target non-coding genes and regulatory loci for transcriptional activation. CRISPRa is most commonly achieved by the fusion of dCas9 with the transactivation domains (ADs) of transcription factors (TFs), which promote transcription by, in turn, recruiting transcriptional and epigenetic machinery, including general transcription factors, the Mediator complex, and chromatin-modifying enzymes. The first generation of CRISPRa vectors used several copies of an 11 amino acid peptide representing the minimal activation domain (AD) of the herpes simplex virus type 1 TF virion protein 16 (VP16)3,5,7. More potent second-generation activators relied on recruiting additional ADs4,6,8,9,10, either by fusion to dCas9 or through bacteriophage RNA-aptamers engineered into the scaffold portion of the sgRNA, or both. Among the most potent and commonly used CRISPRa approaches is the synergistic activation mediator (SAM) system4,10 (Fig. 1a). In the SAM system, dCas9 is fused to four copies of the VP16 minimal AD (VP64) and loaded with an aptamer-modified sgRNA which in turn recruits the MS2 or PP7 bacteriophage coat protein (MCP/PCP)-fused ADs of p65 or HSF1. We will abbreviate MCP or PCP-fused p65AD-HSF1AD synthetic transcriptional activators as MPH and PPH here. The original SAM system consists of 3 lentiviral vectors (LVs)4, expressing dCas9-VP64, MPH, and the aptamer-modified sgRNA, respectively. A subsequent version of SAM aimed to improve the titer of the original MPH-encoding LV and reduce the number of required LVs to two vectors11 by combining a PPH activator protein and an aptamer-modified sgRNA in a single vector (pXPR_502, Fig. 1b). Other systems rely on dCas9 or MCP fusions with several viral or cellular ADs, including VPR (composed of VP64, and p65 and Epstein-Barr virus RTA ADs), and the recently reported NFZ/NZF (composed of compact ADs from NCOA3, FOXO3, and ZNF473 in two different configurations)12, MSN/NMS (composed of ADs from MRTF-A and STAT1, and an engineered AD from NRF2 called eNRF2, in two different configurations)13, and eN3x9 (including eNRF2 and three 9 amino acid ADs from MRTF-B and MYOCD)13. In a conceptually different approach14, with potentially distinct target preferences15, CRISPRa is achieved through dCas9- or sgRNA-mediated recruitment of histone or DNA-modifying enzymatic domains, such as the catalytic histone acetyltransferase (HAT) core domains of the human E1A-associated protein p30016,17 or CBP15,18,19.Fig. 1: Published p65AD-HSF1AD-expressing LVs have low titers and result in lower-than-expected outgrowth after transduction.a Schematic of the SAM CRISPRa system. sgRNA in orange, SL: stem-loop. b Schematic of pXPR_502, not to scale, showing Rous sarcoma virus (RSV)-human immunodeficiency virus hybrid 5′-long terminal repeat (RSV/5′LTR), packaging signal (Ψ), Rev response element (RRE), the U6 promoter, sgRNA location, central polypurine tract (cPPT), human PGK promoter (hPGK), Kozak sequence (K), nuclear localization signals (NLS), PCP, p65AD and HSF1AD coding sequences, separated from a puromycin resistance cassette (PuroR) by a T2A ribosomal skipping peptide, self-inactivating 3′-LTR (SIN 3′LTR). The number at left indicates genome size in kb. c Schematic of pLC-ZsGreen-P2A-Puro, not to scale. Abbreviations as in (b), except for CMV promoter (CMV), ZsGreen coding sequence, and Woodchuck Hepatitis Virus posttranscriptional regulatory element (WPRE). The number at left indicates genome size in kb. d RNA titers of n = 3 independent lentiviral stocks from pLC-ZsGreen-P2A-Puro, or pXPR_502 expressing sgAAVS1 (AAVS1), Calabrese Set A (Cala-A), or sgCRBN-a1 (CRBN). All titers were significantly different from the ZsGreen control, determined using One-Way ANOVA with Tukey’s multiple comparison test. e Functional titers in parental BC-3 of the n = 3 independent LV stocks from (d). All titers were significantly different from the ZsGreen control, determined using One-Way ANOVA with Tukey’s multiple comparison test. f Growth curve analyses of pXPR_502-transduced BC-3/dCas9-VP64. Cumulative live cell counts relative to pLC-ZsGreen-P2A-Puro (ZsGreen)-control transduced cells. Cells were transduced at MOI 0.3, based on either functional (F) or RNA (R) titers. 3 independent repeats, using the 3-LV preps from (d, e). All values differed significantly from the ZsGreen control, determined using One-Way ANOVA with Tukey’s multiple comparison test. n = 3 independent repeats. g Fold increases over the previous passage on days 6, 9, and 12, from the experiments in (f), show that normally proliferating pXPR_502-transduced cells can be grown out 9 days after transduction. Values were normalized to the untransduced and unselected control samples (NT) and differed significantly from NT at each time point unless specified by ns, determined using One-Way ANOVA with Tukey’s multiple comparison test. h Western Blot analyses of CRBN, PPH, and GAPDH in representative lysates taken on day 3 or 12 after transduction from samples shown in (f, g). Endogenous HSF1 is marked “HSF1”. Molecular weight (kDa) markers are at left. For quantification over replicates, see (i). i Quantification of results as shown in (h). Data from 5 (day 3) or 2 (day 12) independent repeats. *** denotes adj. p