Transcriptional Activity of GAL4 BD-NF-kB p65 vs. GAL4 BD-VP16 Fusion Proteins Panel A: Schematic depiction of GAL4 BD-NF-kB p65 and GAL4 BD-VP16 fusion proteins. Tetracycline-Controlled Transcriptional Activation is a method of inducible gene expression where transcription is reversibly turned on or off in the presence of the. Figure 1. CRISPR-on activates exogenous transgenes. (A) Schematic of the dCas9VP48-mediated transgene activation in HeLa cells. dCas9VP48 was generated by fusing.
Cell Research - Multiplexed activation of endogenous genes by CRISPR- on, an RNA- guided transcriptional activator system. Cell Research (2. Aug 2. 01. 3Albert W Cheng. Haoyi Wang. 1,*, Hui Yang. Linyu Shi. 1, Yarden Katz. Thorold W Theunissen. Sudharshan Rangarajan.
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CRISPR interference (CRISPRi) is a genetic perturbation technique that allows for sequence-specific repression or activation of gene expression in prokaryotic and. Abstract. A recessive mutation hsi2 of Arabidopsis (Arabidopsis thaliana) expressing luciferase (LUC) under control of a short promoter derived from a sweet potato.
Chikdu S Shivalila. Daniel B Dadon. 1,4 and Rudolf Jaenisch.
Top of page. Introduction. Gene expression is strictly controlled in many biol- ogical processes, such as development and diseases.
Transcription factors regulate gene expression by binding to specific DNA sequences at the enhancer and promoter regions of target genes, and modulate transcription through their effector domains. Based on the same principle, artificial transcription factors (ATFs) have been generated by fusing various functional domains to a DNA binding domain engineered to bind to the genes of interest, thereby modulating their expression. The capability of regulating endogenous gene expression using ATFs may facilitate the study of the transcriptional network underlying complex biological processes and provide new therapeutic options for diseases. Significant efforts and progress have been made to engineer DNA binding domains with defined specificities. The decipherment of the “code” of DNA binding specificity of zinc finger proteins and transcription activator- like effectors (TALE) has led to the rational design of DNA binding domains to recognize specific nucleotides with certain probability 4,5,6,7,8,9,1. However, binding specificity of these ATFs is usually degenerate, can be difficult to predict and the complex and time- consuming design and generation limits their applications. To study the transcriptional network in a systematic manner, regulating multiple endogenous genes is required, prompting the development of efficient technology for simultaneous regulation of multiple endogenous genes.
CRISPR (clustered regularly interspaced short palin- dromic repeat) and Cas (CRISPR- associated) proteins are utilized by bacteria and archea to defend against viral pathogens. Because the binding of Cas protein is guided by the simple base- pair complementarities between the engineered single guide RNA (sg. RNA) and a target genomic DNA sequence, Cas. RNAs. 13,1. 4,1. 5,1. A recent study described the CRISPRi (CRISPR interference) system, in which the nuclease- deficient d. Cas. 9 (D1. 0A; H8.
A) proteins blocked the transcription apparatus when directed to promoters or gene bodies in bacteria. A subsequent study demonstrated a more efficient gene repression in eukaryotes by d. Cas. 9 fused with a transcription repression domain or exogenous transgene activation when fused with an activation domain. Two most recent studies showed single endogenous gene activation using d.
Cas. 9- based activators. To what extent multiple endogenous genes could be regulated simultaneously has not been explored. In this study we report the generation of an RNA- programmable CRISPR- on system, which enables the simultaneous activation of multiple endogenous genes with a defined stoichiometry. Top of page. Results. Fusion of nuclease- deficient Cas. RNA- programmable transcription factor. To generate a CRISPR/Cas- based transcription activator (CRISPR- on), we introduced the H8.
A mutation in the human codon- optimized Cas. D1. 0A) nickase. 14 to create a nuclease- deficient d. Cas. 9 (H8. 40. A; D1.
A) and fused a 3× minimal VP1. VP4. 8) to its C- terminus (d.
Cas. 9VP4. 8) (Figure 1. A). We first tested d. Cas. 9VP4. 8 in human He. La cells carrying integrated td. Tomato reporter transgene under the control of a Tetracycline- inducible promoter composed of seven copies of rt. TA binding sites and a CMV minimal promoter (Tet.
O: :td. Tomato). As a positive control, these cells constitutively expressed the rt. TA transactivator that induces td.
Tomato expression upon doxycycline treatment (Figure 1. B column ii). Transient transfection of d. Cas. 9VP4. 8 with sg. RNA complementary to rt. TA binding site (sg.
Tet. O) activated the Tet. O: :td. Tomato reporter in the absence of doxycycline at almost the same efficiency as the positive control (Figure 1.
B column iv). Transfection of d. Cas. 9VP4. 8 without sg. RNA did not activate td. Tomato expression (Figure 1. B column iii). Activation of a Tet. O: :td. Tomato reporter lasted for about two weeks but became weak afterwards (Supplementary information, Figure S1). Similarly, co- expression of d.
Cas. 9VP4. 8 with sg. Tet. O activated the td. Tomato transgene in mouse NIH3. T3 cells carrying an integrated Tet. O: :td. Tomato reporter (Supplementary information, Figure S2. B column iv), while expression of d.
Cas. 9VP4. 8 alone did not activate td. Tomato expression (Supplementary information, Figure S2. B column iii). These results indicate that CRISPR- on activates a transgene reporter robustly in human and mouse cells to a similar level to rt.
TA in the presence of doxycycline and that the binding of d. Cas. 9VP4. 8 to the Tet. O promoter is strictly dependent on sg. Tet. O. The higher fraction of fluorescent He. La cells as compared to that in NIH3. T3 cells is likely due to higher transfection efficiency.
CRISPR- on activates exogenous transgenes. A) Schematic of the d. Cas. 9VP4. 8- mediated transgene activation in He. La cells. d. Cas. VP4. 8 was generated by fusing d.
Cas. 9 (indicated by black circle) to VP4. RNA complementary to rt. TA binding site is indicated by small hairpin labeled sg. Tet. O. (B) d. Cas.
VP4. 8 activates Tet. O: :td. Tomato transgene in He. La cells. Upper panel, phase contrast picture of transfected cells; middle panel, td.
Tomato signal using fluorescent microscopy; bottom panel, FACS analysis of transfected cells. Column i, cells transfected with GFP plasmid; column ii, cells treated with doxycycline; column iii, cells transfected with d. Cas. 9VP4. 8 only; column iv, cells transfected with d. Cas. 9VP4. 8 and sg.
Tet. O. Cells were transfected with the indicated plasmids and 4. Tomato expression.
C) Schematic of the d. Cas. 9VP4. 8- mediated reporter activation in early mouse embryos. Cas. 9VP4. 8, Nanog: :EGFP vector, and 7 sg. RNAs targeting Nanog promoter were co- injected into mouse zygotes and cultured into blastocyst stage.
D) d. Cas. 9VP4. 8/sg. RNA can activate gene in vivo. Left panel, embryos injected with d. Cas. 9VP4. 8 and Nanog: :EGFP vector; right panel, embryos injected with d. Cas. 9VP4. 8, Nanog: :EGFP vector and sg. RNAs targeting Nanog promoter.
Embryos two, three, four days post- injection were shown. Full figure and legend (1. K)We tested whether CRISPR- on can activate a single- copy transgene in mouse embryonic stem cells (m.
ESCs). For this, d. Cas. 9VP4. 8 was co- transfected with sg. Tet. O into KH2. MSI1 ESCs carrying a Tet- inducible Musashi. MSI1) transgene at the Col. A1 locus and the rt.
TA- M2 in the Rosa. Supplementary information, Figure S3). Transient transfection of d. Cas. 9VP4. 8 alone did not activate MSI1 expression (Supplementary information, Figure S3 Lane 1), while co- transfection of d. Cas. 9VP4. 8 with sg.
Tet. O or addition of doxycycline (positive control) activated MSI1 expression (Supplementary information, Figure S3 Lane 2 and 7). Neither expression of d.
Cas. 9VP4. 8 with a mutant Tet. O sg. RNA (sg. Tet. O- mut) carrying mismatches to the Tet. O binding sites (Supplementary information, Figure S3 Lane 3) nor expression of sg. Tet. O with d. Cas. MSI1 expression (Supplementary information, Figure S3 Lane 4).
To further characterize the system, we transfected HEK2. T/Tet. O: :td. Tomato cells with d. Cas. 9 activator and a serial titration of sg. RNAs (Supplementary information, Figure S4). We observed a near- linear relationship between the amount of sg. Tet. O transfected and the mean fluorescence by FACS (Supplementary information, Figure S4.
B), indicating that the level of gene activation could be controlled precisely by using CRISPR- on. To test whether CRISPR- on can activate genes in vivo, we co- injected a Nanog: :EGFP construct containing a 1 kb promoter and 5′ UTR of Nanog into mouse zygotes with the d.
Cas. 9VP4. 8 plasmid and seven different sg. RNAs (sg. Nanog- 1~7) targeting the mouse Nanog promoter (Figure 1. C and 1. D). As a control, the Nanog: :EGFP construct was co- injected with d.
Cas. 9VP4. 8 plasmid only. Two days after injection, a GFP signal was detected in 4- cell embryos by fluorescence microscopy and higher GFP expression was observed in morulae and blastocysts on day 3 and day 4, whereas no GFP signal was observed in control embryos injected only with the Nanog: :EGFP construct and d. Cas. 9VP4. 8 plasmid. Although Nanog has been reported to be expressed in cleavage stage embryos. Nanog: :EGFP reporter construct used does not include all necessary elements for Nanog expression in the embryo. Thus, the results shown in Figure 1. D demonstrate that the d.
Cas. 9VP4. 8/sg. Nanogs activator system can specifically activate a GFP transgene by targeting upstream promoter sequences in mouse embryos. Activation of endogenous genes. Having established that the CRISPR- on system can activate reporter transgenes, we designed sg. RNAs targeting the endogenous human IL1. RN gene and tested their transactivation activity in HEK2. T cells. To identify the binding sites most efficient for gene induction, six sg. RNAs were designed to span the 1 kb IL1.
RN promoter (Supplementary information, Figure S5). Initially, we transfected d. Cas. 9VP4. 8 with all 6 sg. RNAs, but failed to induce IL1.
RN gene expression (Supplementary information, Figure S5). To test whether a stronger activation domain can activate IL1. RN, we fused a VP1.
VP1. 6 motifs with d. Cas. 9 to generate d. Cas. 9VP1. 60 (Figure 2. A). When co- transfected with multiple but not single sg. RNAs, d. Cas. 9VP1. IL1. RN (Figure 2.
B and 2. C). Transduction of three proximal sg. RNAs (sg. IL1. RN1~3) activated IL1. RN by approximately 6- fold, whereas the three distal sg. RNAs (sg. IL1. RN4~6) did not induce robust induction. Addition of sg. RNA4~6 to the proximal sg. RNAs (sg. IL1. RN1~3) did not significantly augment the expression (Figure 2.