Since sgRNAs are usually expressed by RNA polymerase III, which limits the length of the RNA domain that can be inserted, CRISPR-Display incorporates RNA polymerase II to permit expression of longer transcripts (~ 80-250 nucleotides) to overcome this limitation. CRISPR-Display can therefore add larger RNA domains, like natural and lncRNA domains, without affecting dCas9 localization. The sgRNA is engineered with an aptameric accessory RNA domain in the sequence outside of the targeting sequence. In the development of the technique, five model cofactors with different topology constructs were used: TOP1-4 and INT with an accessory domain (P4-P6 domain) at different positions, including the 5’ and 3’ end and internally within the sgRNA. Each domain contained a stem-loop that can be recognized by a PP7 bacteriophage coat protein. The complex was delivered into mammalian cells (HEK293FT cells) by a lentiviral vector. 448x448px| Transcription activator assay used to verify sgRNA/Cas9 complex targeting activity, and the proper integrity of the added RNA module. Direct activation: The transcription activator, VP64, is fused to the Cas9 protein, so proper targeting of the complex results in reporter gene activation. Bridged activation: VP64 is fused to PP7, which recognizes and binds a sequence in the RNA module when the RNA module is properly folded. To ensure that the attached RNA module both retains targeting functionality as well as the resulting complex drive transcriptional activation at a specific site of interest, transient reporter gene expression of luciferase and fluorescent protein was measured. Two variations of such a transcription activator assay was performed; directly with a dCas9 fused to a transcriptional activator/repressor (VP64, a factor known to enhance gene expression) (Direct activation) or indirectly where the transcriptional activator is fused to an RNA binding protein module on the sgRNA (Bridged activation). Reporter gene activation through direct activation imply the sgRNA variant binds and targets dCas9 efficiently. All the five topologies showed direct activation except TOP3 and TOP4, which showed reduced activity. Bridged activation indicates that the fused RNA accessory domain is intact in mature dCas9 complexes. Bridged activation was observed with TOP1, TOP3 and INT. The results were recapitulated at endogenous loci by targeting minimal sgRNA and selective expanded topologies (TOP1 and INT) to human ASCL1, IL1RN, NTF3 and TTN promoters. Direct and bridged activation were observed by qRT-PCR for each construct proving that CRISP-Disp allows deployment of large RNA domains to genomic loci. This was also tested with natural lncRNA domains by building Pol II-driven TOP1 and INT constructs fused with human lncRNA domains. lncRNAs used had lengths between ~ 90-4800 nt, and included the NoRC-binding pRNA, three enhancer-transcribed RNAs (eRNAs) FALEC, TRERNA1 and ncRNA-a3), Xist A-repeat (RepA), and the 4,799-nt transcriptional activator HOTTIP. While all the constructs showed significant direct activation, it decreased with increasing lncRNA-sgRNA length. These lncRNA domains could regulate the reporters independent of dCas9 with pRNA and RepA repressing the GLuc reporter expression (repressors) and TRERNA1, ncRNA-a3 and HOTTIP inducing activation (activators), but were properly targeted to an ectopic location of interest by using the CRISP-Disp system. Applications CRISPR-Display allows for previously unavailable studies of lncRNA functionality, artificial ncRNA functionalization, recruitment of endogenous and engineered proteins to genomic loci, and locus affinity tagging for cell imaging. 629x629px| CRISPR-Display allows addition of RNA modules to the sgRNA for a range of functionalities, such as ectopic lncRNA localization, recruitment of endogenous or engineered RNA binding proteins for gene regulation, or affinity tagging for live cell imaging. The functions can be performed simultaneous in the same cell. Affinity tagging for live cell imaging One example of artificial ncRNA functionalization is incorporating RNA domains recognized by specific antibodies to the sgRNA. CRISPR-Display can target the sgRNA with a particular epitope sequence to various loci, and fluorescently tagged antibodies can be used to image the locus, showing its localization in the nucleus, and possible interactions with other tagged proteins or genomic loci.
Since sgRNAs are usually expressed by RNA polymerase III, which limits the length of the RNA domain that can be inserted, CRISPR-Display incorporates RNA polymerase II to permit expression of longer transcripts (~ 80–250 nucleotides) to overcome this limitation. CRISPR-Display can therefore add larger RNA domains, like natural and lncRNA domains, without affecting dCas9 localization. The sgRNA is engineered with an aptameric accessory RNA domain in the sequence outside of the targeting sequence. In the development of the technique, five model cofactors with different topology constructs were used: TOP1-4 and INT with an accessory domain (P4-P6 domain) at different positions, including the 5’ and 3’ end and internally within the sgRNA. Each domain contained a stem-loop that can be recognized by a PP7 bacteriophage coat protein. The complex was delivered into mammalian cells (HEK293FT cells) by a lentiviral vector. Transcription activator assay used to verify sgRNA/Cas9 complex targeting activity, and the proper integrity of the added RNA module. Direct activation: The transcription activator, VP64, is fused to the Cas9 protein, so proper targeting of the complex results in reporter gene activation. Bridged activation: VP64 is fused to PP7, which recognizes and binds a sequence in the RNA module when the RNA module is properly folded. To ensure that the attached RNA module both retains targeting functionality as well as the resulting complex drive transcriptional activation at a specific site of interest, transient reporter gene expression of luciferase and fluorescent protein was measured. Two variations of such a transcription activator assay was performed; directly with a dCas9 fused to a transcriptional activator/repressor (VP64, a factor known to enhance gene expression) (Direct activation) or indirectly where the transcriptional activator is fused to an RNA binding protein module on the sgRNA (Bridged activation). Reporter gene activation through direct activation imply the sgRNA variant binds and targets dCas9 efficiently. All the five topologies showed direct activation except TOP3 and TOP4, which showed reduced activity. Bridged activation indicates that the fused RNA accessory domain is intact in mature dCas9 complexes. Bridged activation was observed with TOP1, TOP3 and INT. The results were recapitulated at endogenous loci by targeting minimal sgRNA and selective expanded topologies (TOP1 and INT) to human ASCL1, IL1RN, NTF3 and TTN promoters. Direct and bridged activation were observed by qRT-PCR for each construct proving that CRISP-Disp allows deployment of large RNA domains to genomic loci. This was also tested with natural lncRNA domains by building Pol II-driven TOP1 and INT constructs fused with human lncRNA domains. lncRNAs used had lengths between ~ 90–4800 nt, and included the NoRC-binding pRNA, three enhancer-transcribed RNAs (eRNAs) FALEC, TRERNA1 and ncRNA-a3), Xist A-repeat (RepA), and the 4,799-nt transcriptional activator HOTTIP. While all the constructs showed significant direct activation, it decreased with increasing lncRNA-sgRNA length. These lncRNA domains could regulate the reporters independent of dCas9 with pRNA and RepA repressing the GLuc reporter expression (repressors) and TRERNA1, ncRNA-a3 and HOTTIP inducing activation (activators), but were properly targeted to an ectopic location of interest by using the CRISP-Disp system. Applications CRISPR-Display allows for previously unavailable studies of lncRNA functionality, artificial ncRNA functionalization, recruitment of endogenous and engineered proteins to genomic loci, and locus affinity tagging for cell imaging. CRISPR-Display allows addition of RNA modules to the sgRNA for a range of functionalities, such as ectopic lncRNA localization, recruitment of endogenous or engineered RNA binding proteins for gene regulation, or affinity tagging for live cell imaging. The functions can be performed simultaneous in the same cell. Affinity tagging for live cell imaging One example of artificial ncRNA functionalization is incorporating RNA domains recognized by specific antibodies to the sgRNA. CRISPR-Display can target the sgRNA with a particular epitope sequence to various loci, and fluorescently tagged antibodies can be used to image the locus, showing its localization in the nucleus, and possible interactions with other tagged proteins or genomic loci.
R
80-250
80–250
D
448x448px|
R
90-4800
90–4800
D
629x629px|