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Evaluation of sgRNA target sites for CRISPR-mediated repression of TP53.

Lawhorn IE, Ferreira JP, Wang CL - PLoS ONE (2014)

Bottom Line: The sites spanned a 200-kb distance, which included the promoter, transcript sequence, and regions flanking the endogenous human TP53 gene.We showed that repression up to 86% can be achieved with dCas9 alone (i.e., without use of the KRAB domain) by targeting the complex to sites near the TP53 transcriptional start site.This work demonstrates that efficient transcriptional repression of endogenous human genes can be achieved by the targeted delivery of dCas9.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, Stanford University, Stanford, California, United States of America.

ABSTRACT
The CRISPR (clustered regularly interspaced short palindromic repeats) platform has been developed as a general method to direct proteins of interest to gene targets. While the native CRISPR system delivers a nuclease that cleaves and potentially mutates target genes, researchers have recently employed catalytically inactive CRISPR-associated 9 nuclease (dCas9) in order to target and repress genes without DNA cleavage or mutagenesis. With the intent of improving repression efficiency in mammalian cells, researchers have also fused dCas9 with a KRAB repressor domain. Here, we evaluated different genomic sgRNA targeting sites for repression of TP53. The sites spanned a 200-kb distance, which included the promoter, transcript sequence, and regions flanking the endogenous human TP53 gene. We showed that repression up to 86% can be achieved with dCas9 alone (i.e., without use of the KRAB domain) by targeting the complex to sites near the TP53 transcriptional start site. This work demonstrates that efficient transcriptional repression of endogenous human genes can be achieved by the targeted delivery of dCas9. Yet, the efficiency of repression strongly depends on the choice of the sgRNA target site.

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Repression of TP53 by targeting dCas9 to the transcriptional start site.(A) HEK 293T cells were co-transfected with dCas9 (left) and sgRNA (right) expression plasmids. Codon-optimized dCas9 was fused to three copies of nuclear localization signal (NLS) and was co-expressed with mCherry fluorescent protein. sgRNA plasmid expresses mBFP and sgRNA off separate promoters. PCMV, CMV promoter; 2A, ribosomal slippage site; PuroR, puromycin resistance gene; IRES, internal ribosome entry site; mBFP, TagBFP fluorescent protein; BlastR, blasticidin resistance gene; PU6, U6 promoter. (B) Locations of sgRNA binding sites in the TP53 promoter and transcribed region (thick line). Each sgRNA is numbered by the distance (bp) from the transcriptional start site. “−”, upstream of +1; “+”, downstream of +1; “K”, one thousand bp; +1, transcriptional start site. Labeled sites above and below the transcribed region indicate sgRNAs targeting the template or non-template DNA strands, respectively. (C, D) Relative expression of TP53 mRNA in cells co-transfected with dCas9 and indicated sgRNA constructs. After three days, cells were (C) directly analyzed by qRT-PCR or (D) sorted for BFP-positive cells, then analyzed by qRT-PCR. Results were normalized, linearly rescaled, and calculated for mean fold change (n = 3)±95% confidence interval, relative to Scramble1 negative control sgRNA. *P<0.01 compared to non-targeted sgRNA control by paired, one-sided t-test. (E) Immunoblot from HEK 293T cell lysate three days post-transfection with shRNA against TP53, dCas9 and sgRNAs against TP53 (e.g., dCas9/−T36), dCas9 and non-targeted sgRNA (dCas9/Scramble1), other control constructs (GFP expression vector, which served as non-specific vector control, or pSuper without any shRNA encoded), or mock (PEI only). See Figure 2E for location of sgRNA +T111. pSuper constructs were co-transfected with GFP-IRES-mCherry-2A-Puro expression constructs for selection purposes and experimental consistency (pSuper/GFP, pSuper-p53/GFP). Protein ladder (lane 2 from left) is not visible. See Figure S5 for uncropped immunoblots.
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pone-0113232-g001: Repression of TP53 by targeting dCas9 to the transcriptional start site.(A) HEK 293T cells were co-transfected with dCas9 (left) and sgRNA (right) expression plasmids. Codon-optimized dCas9 was fused to three copies of nuclear localization signal (NLS) and was co-expressed with mCherry fluorescent protein. sgRNA plasmid expresses mBFP and sgRNA off separate promoters. PCMV, CMV promoter; 2A, ribosomal slippage site; PuroR, puromycin resistance gene; IRES, internal ribosome entry site; mBFP, TagBFP fluorescent protein; BlastR, blasticidin resistance gene; PU6, U6 promoter. (B) Locations of sgRNA binding sites in the TP53 promoter and transcribed region (thick line). Each sgRNA is numbered by the distance (bp) from the transcriptional start site. “−”, upstream of +1; “+”, downstream of +1; “K”, one thousand bp; +1, transcriptional start site. Labeled sites above and below the transcribed region indicate sgRNAs targeting the template or non-template DNA strands, respectively. (C, D) Relative expression of TP53 mRNA in cells co-transfected with dCas9 and indicated sgRNA constructs. After three days, cells were (C) directly analyzed by qRT-PCR or (D) sorted for BFP-positive cells, then analyzed by qRT-PCR. Results were normalized, linearly rescaled, and calculated for mean fold change (n = 3)±95% confidence interval, relative to Scramble1 negative control sgRNA. *P<0.01 compared to non-targeted sgRNA control by paired, one-sided t-test. (E) Immunoblot from HEK 293T cell lysate three days post-transfection with shRNA against TP53, dCas9 and sgRNAs against TP53 (e.g., dCas9/−T36), dCas9 and non-targeted sgRNA (dCas9/Scramble1), other control constructs (GFP expression vector, which served as non-specific vector control, or pSuper without any shRNA encoded), or mock (PEI only). See Figure 2E for location of sgRNA +T111. pSuper constructs were co-transfected with GFP-IRES-mCherry-2A-Puro expression constructs for selection purposes and experimental consistency (pSuper/GFP, pSuper-p53/GFP). Protein ladder (lane 2 from left) is not visible. See Figure S5 for uncropped immunoblots.

Mentions: Since dCas9-KRAB had little added benefit over dCas9 for repression of TP53 and there was significant repression when targeting sites near the TSS, we further explored the positional effect of targeting dCas9 to sites closer to the TP53 gene itself. 21 sgRNAs were designed to target sequences on the template and non-template strands of the promoter and transcribed region of TP53 (Table S1 and Figure 1A and B). In bacteria, only sgRNAs binding to the non-template strand could enable repression of a fluorescent protein [15] although that finding has not been replicated in mammalian cells. The sgRNAs were co-expressed with dCas9 in transfected and puromycin-selected HEK 293T cells and directly analyzed by qRT-PCR. As before, the −T36 target site was shown to significantly repress TP53 expression at the mRNA level (P<0.01, Figure 1C) as compared to cells expressing either non-targeted control. Because transfection efficiency varied, we opted to sort for BFP-positive cells after transfection and puromycin selection to enrich for cells expressing both CRISPR constructs. Of the subset of sgRNAs tested, three repressed TP53 transcriptional expression (Figure 1D), all located within a 200-bp region of the TSS. sgRNA −T36 induced approximately 86% TP53 mRNA repression, a level comparable to shRNA knock-down with pSuper-p53 construct [26] (Figure S3C). Repression using this target site was also consistently observed at the protein level in unsorted, selected cells (Figure 1E) for three independent experiments. However, when targeting the +T110 and +T111 sites and analyzing protein levels by immunoblotting, not only were the magnitudes of repression lower than −T36 but they were also less reproducible over those same experiments. Additionally, the targeting of sites +T27 and +T110 repressed TP53 transcription expression. We did not observe significant repression when targeting the template or non-template strands of DNA in the transcribed region. It should be noted that first exon of TP53 overlaps with exon 1α of WRAP53[38], [39] which is the first exon of the WRAP53α isoform—a known anti-sense transcript of TP53 (Figure S3A). We observed repression of the WRAP53α isoform in cells transfected with dCas9 and the −T36 sgRNA but no similar repression of total WRAP53 expression from all three isoforms (Figure S3B); we did not see a similar effect with pSuper-p53 shRNA transfection (Figure S3C). Finally, we note that for the many sgRNAs in our study that did not generate appreciable repression of TP53, we cannot rule out that these sgRNAs were somehow incapable of mediating the binding of dCas9 to the DNA.


Evaluation of sgRNA target sites for CRISPR-mediated repression of TP53.

Lawhorn IE, Ferreira JP, Wang CL - PLoS ONE (2014)

Repression of TP53 by targeting dCas9 to the transcriptional start site.(A) HEK 293T cells were co-transfected with dCas9 (left) and sgRNA (right) expression plasmids. Codon-optimized dCas9 was fused to three copies of nuclear localization signal (NLS) and was co-expressed with mCherry fluorescent protein. sgRNA plasmid expresses mBFP and sgRNA off separate promoters. PCMV, CMV promoter; 2A, ribosomal slippage site; PuroR, puromycin resistance gene; IRES, internal ribosome entry site; mBFP, TagBFP fluorescent protein; BlastR, blasticidin resistance gene; PU6, U6 promoter. (B) Locations of sgRNA binding sites in the TP53 promoter and transcribed region (thick line). Each sgRNA is numbered by the distance (bp) from the transcriptional start site. “−”, upstream of +1; “+”, downstream of +1; “K”, one thousand bp; +1, transcriptional start site. Labeled sites above and below the transcribed region indicate sgRNAs targeting the template or non-template DNA strands, respectively. (C, D) Relative expression of TP53 mRNA in cells co-transfected with dCas9 and indicated sgRNA constructs. After three days, cells were (C) directly analyzed by qRT-PCR or (D) sorted for BFP-positive cells, then analyzed by qRT-PCR. Results were normalized, linearly rescaled, and calculated for mean fold change (n = 3)±95% confidence interval, relative to Scramble1 negative control sgRNA. *P<0.01 compared to non-targeted sgRNA control by paired, one-sided t-test. (E) Immunoblot from HEK 293T cell lysate three days post-transfection with shRNA against TP53, dCas9 and sgRNAs against TP53 (e.g., dCas9/−T36), dCas9 and non-targeted sgRNA (dCas9/Scramble1), other control constructs (GFP expression vector, which served as non-specific vector control, or pSuper without any shRNA encoded), or mock (PEI only). See Figure 2E for location of sgRNA +T111. pSuper constructs were co-transfected with GFP-IRES-mCherry-2A-Puro expression constructs for selection purposes and experimental consistency (pSuper/GFP, pSuper-p53/GFP). Protein ladder (lane 2 from left) is not visible. See Figure S5 for uncropped immunoblots.
© Copyright Policy
Related In: Results  -  Collection

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pone-0113232-g001: Repression of TP53 by targeting dCas9 to the transcriptional start site.(A) HEK 293T cells were co-transfected with dCas9 (left) and sgRNA (right) expression plasmids. Codon-optimized dCas9 was fused to three copies of nuclear localization signal (NLS) and was co-expressed with mCherry fluorescent protein. sgRNA plasmid expresses mBFP and sgRNA off separate promoters. PCMV, CMV promoter; 2A, ribosomal slippage site; PuroR, puromycin resistance gene; IRES, internal ribosome entry site; mBFP, TagBFP fluorescent protein; BlastR, blasticidin resistance gene; PU6, U6 promoter. (B) Locations of sgRNA binding sites in the TP53 promoter and transcribed region (thick line). Each sgRNA is numbered by the distance (bp) from the transcriptional start site. “−”, upstream of +1; “+”, downstream of +1; “K”, one thousand bp; +1, transcriptional start site. Labeled sites above and below the transcribed region indicate sgRNAs targeting the template or non-template DNA strands, respectively. (C, D) Relative expression of TP53 mRNA in cells co-transfected with dCas9 and indicated sgRNA constructs. After three days, cells were (C) directly analyzed by qRT-PCR or (D) sorted for BFP-positive cells, then analyzed by qRT-PCR. Results were normalized, linearly rescaled, and calculated for mean fold change (n = 3)±95% confidence interval, relative to Scramble1 negative control sgRNA. *P<0.01 compared to non-targeted sgRNA control by paired, one-sided t-test. (E) Immunoblot from HEK 293T cell lysate three days post-transfection with shRNA against TP53, dCas9 and sgRNAs against TP53 (e.g., dCas9/−T36), dCas9 and non-targeted sgRNA (dCas9/Scramble1), other control constructs (GFP expression vector, which served as non-specific vector control, or pSuper without any shRNA encoded), or mock (PEI only). See Figure 2E for location of sgRNA +T111. pSuper constructs were co-transfected with GFP-IRES-mCherry-2A-Puro expression constructs for selection purposes and experimental consistency (pSuper/GFP, pSuper-p53/GFP). Protein ladder (lane 2 from left) is not visible. See Figure S5 for uncropped immunoblots.
Mentions: Since dCas9-KRAB had little added benefit over dCas9 for repression of TP53 and there was significant repression when targeting sites near the TSS, we further explored the positional effect of targeting dCas9 to sites closer to the TP53 gene itself. 21 sgRNAs were designed to target sequences on the template and non-template strands of the promoter and transcribed region of TP53 (Table S1 and Figure 1A and B). In bacteria, only sgRNAs binding to the non-template strand could enable repression of a fluorescent protein [15] although that finding has not been replicated in mammalian cells. The sgRNAs were co-expressed with dCas9 in transfected and puromycin-selected HEK 293T cells and directly analyzed by qRT-PCR. As before, the −T36 target site was shown to significantly repress TP53 expression at the mRNA level (P<0.01, Figure 1C) as compared to cells expressing either non-targeted control. Because transfection efficiency varied, we opted to sort for BFP-positive cells after transfection and puromycin selection to enrich for cells expressing both CRISPR constructs. Of the subset of sgRNAs tested, three repressed TP53 transcriptional expression (Figure 1D), all located within a 200-bp region of the TSS. sgRNA −T36 induced approximately 86% TP53 mRNA repression, a level comparable to shRNA knock-down with pSuper-p53 construct [26] (Figure S3C). Repression using this target site was also consistently observed at the protein level in unsorted, selected cells (Figure 1E) for three independent experiments. However, when targeting the +T110 and +T111 sites and analyzing protein levels by immunoblotting, not only were the magnitudes of repression lower than −T36 but they were also less reproducible over those same experiments. Additionally, the targeting of sites +T27 and +T110 repressed TP53 transcription expression. We did not observe significant repression when targeting the template or non-template strands of DNA in the transcribed region. It should be noted that first exon of TP53 overlaps with exon 1α of WRAP53[38], [39] which is the first exon of the WRAP53α isoform—a known anti-sense transcript of TP53 (Figure S3A). We observed repression of the WRAP53α isoform in cells transfected with dCas9 and the −T36 sgRNA but no similar repression of total WRAP53 expression from all three isoforms (Figure S3B); we did not see a similar effect with pSuper-p53 shRNA transfection (Figure S3C). Finally, we note that for the many sgRNAs in our study that did not generate appreciable repression of TP53, we cannot rule out that these sgRNAs were somehow incapable of mediating the binding of dCas9 to the DNA.

Bottom Line: The sites spanned a 200-kb distance, which included the promoter, transcript sequence, and regions flanking the endogenous human TP53 gene.We showed that repression up to 86% can be achieved with dCas9 alone (i.e., without use of the KRAB domain) by targeting the complex to sites near the TP53 transcriptional start site.This work demonstrates that efficient transcriptional repression of endogenous human genes can be achieved by the targeted delivery of dCas9.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, Stanford University, Stanford, California, United States of America.

ABSTRACT
The CRISPR (clustered regularly interspaced short palindromic repeats) platform has been developed as a general method to direct proteins of interest to gene targets. While the native CRISPR system delivers a nuclease that cleaves and potentially mutates target genes, researchers have recently employed catalytically inactive CRISPR-associated 9 nuclease (dCas9) in order to target and repress genes without DNA cleavage or mutagenesis. With the intent of improving repression efficiency in mammalian cells, researchers have also fused dCas9 with a KRAB repressor domain. Here, we evaluated different genomic sgRNA targeting sites for repression of TP53. The sites spanned a 200-kb distance, which included the promoter, transcript sequence, and regions flanking the endogenous human TP53 gene. We showed that repression up to 86% can be achieved with dCas9 alone (i.e., without use of the KRAB domain) by targeting the complex to sites near the TP53 transcriptional start site. This work demonstrates that efficient transcriptional repression of endogenous human genes can be achieved by the targeted delivery of dCas9. Yet, the efficiency of repression strongly depends on the choice of the sgRNA target site.

Show MeSH