A potent Cas9-derived gene activator for plant and mammalian cells

Abstract

Overexpression of complementary DNA (cDNA) represents the most commonly used gain-of-function approach for interrogating gene functions and for manipulating biological traits. However, this approach is challenging and inefficient for multigene expression due to increased labor for cloning, limited vector capacity, requirement of multiple promoters and terminators, and variable transgene expression levels. Synthetic transcriptional activators provide a promising alternative strategy for gene activation by tethering an autonomous transcription activation domain (TAD) to an intended gene promoter at endogenous genomic locus through a programmable DNA-binding module. Among the known custom DNA-binding modules, the nuclease-dead Streptococcus pyogenes Cas9 (dCas9) protein, which recognizes a specific DNA target through base pairing between a synthetic guide RNA (sgRNA) and DNA, outperforms zinc finger proteins (ZFPs) and transcription activator-like effectors (TALEs), both of which target through protein-DNA interactions1. Recently, three potent dCas9-based transcriptional activation systems, namely VPR, SAM, and Suntag, have been developed for animal cells2–6. However, an efficient dCas9-based transcriptional activation platform is still lacking for plant cells7–9. Here, we developed a new potent dCas9-TAD named dCas9-TV through plant cell-based screens, which confers far stronger transcriptional activation of a single or multiple target genes than the routinely used dCas9-VP64 activator in both plant and mammalian cells.

Targeted Transcriptional Activation in Plants Using a Potent Dead Cas9–Derived Synthetic Gene Activator

“…An inverse correlation between the extent of gene induction and the basal transcript level of target genes was observed in mammalian cells (Konermann et al., ). Likewise, we observed the same trend in plant cells, i.e., genes with higher basal transcript levels are less inducible by dCas9‐TV (Li et al., ). This is presumably because dCas9‐TV or other potent dCas9 gene activators have to compete with endogenous transcriptional activators in the case of genes that are being transcribed at high levels.…”

“…However, not all sgRNA targets located upstream of the TSS confer efficient gene activation. Different sgRNAs with different target sites within the same promoter exhibit great variation in the levels of gene induction (Li et al., ). According to a previous study, the highest level of activation was consistently achieved by targeting dCas9‐derived activators within the –200 bp to –1 bp window upstream of TSSs (Konermann et al., ).…”

“…Transcriptional activation of multiple genes can be achieved by concurrently targeting multiple promoters with a set of sgRNAs and dCas9‐derived gene activators. With the improved dCas9 gene activators, we and others have demonstrated multiplexed activation for ≥3 endogenous genes by co‐expressing multiple sgRNAs, each targeting one gene's promoter (Konermann et al., ; Li et al., ). To obtain multiplexed gene activation in transgenic plants, one can first identify the most efficient sgRNA for each target gene, then insert the corresponding sgRNA expression cassettes into the multiple cloning sites of the pUC119 vector (Fig.…”

Gene disruption through base editing‐induced messenger RNA missplicing in plants

“…S1a). To simultaneously edit OsGL1‐1 and OsNAL1 in rice, the OsU6a:sgRNA‐GL1‐1:TTTTTT and OsU6b:sgRNA‐NAL1:TTTTTT expression cassettes were individually generated through overlapping PCR and were inserted into the pUC119‐RCS (Li Z. et al ., ) cloning vector at Kpn I/ Hind III and Sbf I sites, respectively. A fragment containing these two sgRNA expression cassettes was PCR amplified and cloned into the Hind III‐digested pH‐nCas9‐PBE binary plasmid (Fig.…”

In vivo epigenome editing and transcriptional modulation using CRISPR technology

“…dCas9-KRAB) are usually transient, but epigenetic marks left by targeted epigenetic modifiers can be inherited by the daughter cells (Amabile et al 2016). In addition, dCas9-VPR (Chavez et al 2015) and dCas9-TV (Li et al 2017) are more potent transactivation domains than the dCas9-VP64. dCas9-VPR is the fusion of dCas9 to a tripartite VP64-p65-Rta (VPR) transactivation domain.…”