Controlling mRNA stability and translation with the CRISPR endoribonuclease Csy4

Abstract

The bacterial CRISPR endoribonuclease Csy4 has recently been described as a potential RNA processing tool. Csy4 recognizes substrate RNA through a specific 28-nt hairpin sequence and cleaves at the 3 ′ end of the stem. To further explore applicability in mammalian cells, we introduced this hairpin at various locations in mRNAs derived from reporter transgenes and systematically evaluated the effects of Csy4-mediated processing on transgene expression. Placing the hairpin in the 5 ′ UTR or immediately after the start codon resulted in efficient degradation of target mRNA by Csy4 and knockdown of transgene expression by 20-to 40-fold. When the hairpin was incorporated in the 3 ′ UTR prior to the poly(A) signal, the mRNA was cleaved, but only a modest decrease in transgene expression (∼2.5-fold) was observed. In the absence of a poly(A) tail, Csy4 rescued the target mRNA substrate from degradation, resulting in protein expression, which suggests that the cleaved mRNA was successfully translated. In contrast, neither catalytically inactive (H29A) nor binding-deficient (R115A/R119A) Csy4 mutants were able to exert any of the effects described above. Generation of a similar 3 ′ end by RNase P-mediated cleavage was unable to rescue transgene expression independent of Csy4. These results support the idea that the selective generation of the Csy4/hairpin complex resulting from cleavage of target mRNA might serve as a functional poly(A)/poly(A) binding protein (PABP) surrogate, stabilizing the mRNA and supporting translation. Although the exact mechanism(s) remain to be determined, our studies expand the potential utility of CRISPR nucleases as tools for controlling mRNA stability and translation.

PERSIST: A programmable RNA regulation platform using CRISPR endoRNases

“…We first developed an endoRNase-responsive ON switch using Csy4. The CRISPR endoRNase Csy4 (PaeCas6f) has been used as a translational repressor in mammalian synthetic biology previously [10,49,72] and here we show that it can be repurposed to activate expression when its cleavage site is placed within the PERSIST-ON motif (Supplementary Figure 4). We chose to benchmark the Csy4-responsive PERSIST ON-switch against the routinely-used Tet-On system [31].…”

“…We first set out to develop RNA-level switches that enable turning transgene expression on or off through regulation of transcript degradation. Previous studies have shown that transcript cleavage, for example by miRNA [25] or endoribonucleases [10,49,72], can reduce transgene expression ( Figure 1a, left). Our initial object was to develop an RNA-level ON switch that activates gene expression in response to transcript cleavage.…”

“…Some protein-based RNA-level regulators exist, mainly using translational repressors [56,60,66] and RNA-cleaving Cas proteins [10,1,2,16,59,41,69], but there is still a great need for demonstrated scalability and composability. Also, all of these examples canonically act as only repressor-like "OFF" switches, where a few RNA-based activator-like "ON" switches have been developed [9,10,18,56,3], none of which are composable into circuits. The field currently lacks an RNA-acting activation/repression toolkit that is scalable, robust, modular and composable to replace transcription factors.…”

Biotechnology and synthetic biology approaches for metabolic engineering of bioenergy crops

“…In E. coli , yeast, and mammalian cell studies, effective cleavage at one or more CSY4 cognition sites in one transcript was demonstrated. Because of the broad functionality of CSY4 in these organisms, it can be expected that site‐targeted endoribonucleases can be fashioned to work in plants by adding CSY4's recognition cognition sequence in either the coding sequence or untranslated regulatory regions (Qi et al ., ; Eudes et al ., ; Nissim et al ., ; Borchardt et al ., ). Such an RNase‐based system can be used to generate context‐free genetic elements, resulting in predictable expression regulation of one or multiple genes.…”

CRISPR-Csy4-mediated editing of rotavirus double-stranded RNA genome

“…RNA cleavage activity of the different Csy4 variants was evaluated using an EGFP reporter 127 plasmid having the Cy28 target sequence located immediately after the ATG initiation codon 128 ( Figure 2A) (Borchardt et al, 2015). Csy4-mediated cleavage of the reporter transcript was 129 6 measured by the reduction of EGFP intensity (Borchardt et al, 2015). As shown in Figure 130 2A, Cy28-EGFP levels were strongly but similarly diminished in MA-NSP2-Csy4 and MA-131…”