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RNA-guided CRISPR-Cas technologies for genome-scale investigation of disease processes.

Humphrey SE, Kasinski AL - J Hematol Oncol (2015)

Bottom Line: From its discovery as an adaptive bacterial and archaea immune system, the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has quickly been developed into a powerful and groundbreaking programmable nuclease technology for the global and precise editing of the genome in cells.This system allows for comprehensive unbiased functional studies and is already advancing the field by revealing genes that have previously unknown roles in disease processes.We also explore some of the exciting therapeutic potentials of the CRISPR-Cas technology as well as some of the innovative new uses of this technology beyond genome editing.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Purdue University, 1203 West State Street, West Lafayette, IN, 47907, USA. shumphr@purdue.edu.

ABSTRACT
From its discovery as an adaptive bacterial and archaea immune system, the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has quickly been developed into a powerful and groundbreaking programmable nuclease technology for the global and precise editing of the genome in cells. This system allows for comprehensive unbiased functional studies and is already advancing the field by revealing genes that have previously unknown roles in disease processes. In this review, we examine and compare recently developed CRISPR-Cas platforms for global genome editing and examine the advancements these platforms have made in guide RNA design, guide RNA/Cas9 interaction, on-target specificity, and target sequence selection. We also explore some of the exciting therapeutic potentials of the CRISPR-Cas technology as well as some of the innovative new uses of this technology beyond genome editing.

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Applications for the CRISPR-Cas9 system beyond gene editing. (A) CRISPR-Cas9 as a tool for inhibiting transcriptional activation. sgRNAs can be used to direct the binding of catalytically inactive Cas9 (dCas9) to the promoter regions of genes. Once bound, dCas9 can interfere with transcriptional initiation of the gene and thus inhibit gene expression. (B) CRISPR-Cas9 to promote the transcription of a gene. sgRNAs can be used to direct the binding of a catalytically inactive Cas9 protein fused to a transcriptional activation domain (dCas9/TAD) to the promoter regions of genes. Once bound, dCas9/TAD can promote transcription of the target gene. (C) CRISPR-Cas9 to image various elements of the genome. sgRNAs can be used to direct the binding of catalytically inactive Cas9 fused to enhanced green fluorescent protein (dCas9/EGFP) to various elements of the genome. This technology can be used to image different elements of a chromosome, telomeres in this example, in live cells. Dynamic chromosomal changes during growth and replication can also be imaged.
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Fig3: Applications for the CRISPR-Cas9 system beyond gene editing. (A) CRISPR-Cas9 as a tool for inhibiting transcriptional activation. sgRNAs can be used to direct the binding of catalytically inactive Cas9 (dCas9) to the promoter regions of genes. Once bound, dCas9 can interfere with transcriptional initiation of the gene and thus inhibit gene expression. (B) CRISPR-Cas9 to promote the transcription of a gene. sgRNAs can be used to direct the binding of a catalytically inactive Cas9 protein fused to a transcriptional activation domain (dCas9/TAD) to the promoter regions of genes. Once bound, dCas9/TAD can promote transcription of the target gene. (C) CRISPR-Cas9 to image various elements of the genome. sgRNAs can be used to direct the binding of catalytically inactive Cas9 fused to enhanced green fluorescent protein (dCas9/EGFP) to various elements of the genome. This technology can be used to image different elements of a chromosome, telomeres in this example, in live cells. Dynamic chromosomal changes during growth and replication can also be imaged.

Mentions: Using the CRISPR-Cas system for genome editing is just the beginning of its utility. For example, CRISPR-Cas can be used to regulate the expression of genes. Catalytically inactive Cas9 (dCas9) guided to the promoter region of a gene can repress transcription by interfering with transcriptional elongation (Figure 3A) [59,60]. Transcriptional repression can be enhanced by fusing a transcriptional repression domain to dCas9 [61,62]. Likewise, dCas9 can be fused to a transactivation domain and be used to upregulate the expression of a gene (Figure 3B) [49,63-66].Figure 3


RNA-guided CRISPR-Cas technologies for genome-scale investigation of disease processes.

Humphrey SE, Kasinski AL - J Hematol Oncol (2015)

Applications for the CRISPR-Cas9 system beyond gene editing. (A) CRISPR-Cas9 as a tool for inhibiting transcriptional activation. sgRNAs can be used to direct the binding of catalytically inactive Cas9 (dCas9) to the promoter regions of genes. Once bound, dCas9 can interfere with transcriptional initiation of the gene and thus inhibit gene expression. (B) CRISPR-Cas9 to promote the transcription of a gene. sgRNAs can be used to direct the binding of a catalytically inactive Cas9 protein fused to a transcriptional activation domain (dCas9/TAD) to the promoter regions of genes. Once bound, dCas9/TAD can promote transcription of the target gene. (C) CRISPR-Cas9 to image various elements of the genome. sgRNAs can be used to direct the binding of catalytically inactive Cas9 fused to enhanced green fluorescent protein (dCas9/EGFP) to various elements of the genome. This technology can be used to image different elements of a chromosome, telomeres in this example, in live cells. Dynamic chromosomal changes during growth and replication can also be imaged.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4389696&req=5

Fig3: Applications for the CRISPR-Cas9 system beyond gene editing. (A) CRISPR-Cas9 as a tool for inhibiting transcriptional activation. sgRNAs can be used to direct the binding of catalytically inactive Cas9 (dCas9) to the promoter regions of genes. Once bound, dCas9 can interfere with transcriptional initiation of the gene and thus inhibit gene expression. (B) CRISPR-Cas9 to promote the transcription of a gene. sgRNAs can be used to direct the binding of a catalytically inactive Cas9 protein fused to a transcriptional activation domain (dCas9/TAD) to the promoter regions of genes. Once bound, dCas9/TAD can promote transcription of the target gene. (C) CRISPR-Cas9 to image various elements of the genome. sgRNAs can be used to direct the binding of catalytically inactive Cas9 fused to enhanced green fluorescent protein (dCas9/EGFP) to various elements of the genome. This technology can be used to image different elements of a chromosome, telomeres in this example, in live cells. Dynamic chromosomal changes during growth and replication can also be imaged.
Mentions: Using the CRISPR-Cas system for genome editing is just the beginning of its utility. For example, CRISPR-Cas can be used to regulate the expression of genes. Catalytically inactive Cas9 (dCas9) guided to the promoter region of a gene can repress transcription by interfering with transcriptional elongation (Figure 3A) [59,60]. Transcriptional repression can be enhanced by fusing a transcriptional repression domain to dCas9 [61,62]. Likewise, dCas9 can be fused to a transactivation domain and be used to upregulate the expression of a gene (Figure 3B) [49,63-66].Figure 3

Bottom Line: From its discovery as an adaptive bacterial and archaea immune system, the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has quickly been developed into a powerful and groundbreaking programmable nuclease technology for the global and precise editing of the genome in cells.This system allows for comprehensive unbiased functional studies and is already advancing the field by revealing genes that have previously unknown roles in disease processes.We also explore some of the exciting therapeutic potentials of the CRISPR-Cas technology as well as some of the innovative new uses of this technology beyond genome editing.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Purdue University, 1203 West State Street, West Lafayette, IN, 47907, USA. shumphr@purdue.edu.

ABSTRACT
From its discovery as an adaptive bacterial and archaea immune system, the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has quickly been developed into a powerful and groundbreaking programmable nuclease technology for the global and precise editing of the genome in cells. This system allows for comprehensive unbiased functional studies and is already advancing the field by revealing genes that have previously unknown roles in disease processes. In this review, we examine and compare recently developed CRISPR-Cas platforms for global genome editing and examine the advancements these platforms have made in guide RNA design, guide RNA/Cas9 interaction, on-target specificity, and target sequence selection. We also explore some of the exciting therapeutic potentials of the CRISPR-Cas technology as well as some of the innovative new uses of this technology beyond genome editing.

Show MeSH