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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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Dual CRISPR-Cas technologies that increase nuclease specificity. (A, B) Dimeric CRISPR RNA-guided Fok1 nuclease target sequences consist of two 20 nt half-sites flanked by a protospacer adjacent motif (PAM) sequence in the form of 5′-NGG that are separated by a 14–17 nt spacer sequence. Each half-site is bound by a Cas-9/Fok1 fusion protein. Once bound, the Fok1 domains of two different Cas-9/Fok1 fusion proteins dimerize and introduce a double-stranded break in the spacer sequence. (C) Dual RNA-guided CRISPR-Cas9 nickase system. In this system, two sgRNAs are expressed that each guide a mutant version of Cas9 (Cas9-D10A) (that only nicks one strand of the DNA rather than making a double-stranded cut) to two different sequences that flank the target region. The two Cas9 nickases bind to opposite strands of the DNA nicking both DNA strands flanking the target region. This introduces a site-specific double-stranded break that is then repaired by NHEJ.
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Fig2: Dual CRISPR-Cas technologies that increase nuclease specificity. (A, B) Dimeric CRISPR RNA-guided Fok1 nuclease target sequences consist of two 20 nt half-sites flanked by a protospacer adjacent motif (PAM) sequence in the form of 5′-NGG that are separated by a 14–17 nt spacer sequence. Each half-site is bound by a Cas-9/Fok1 fusion protein. Once bound, the Fok1 domains of two different Cas-9/Fok1 fusion proteins dimerize and introduce a double-stranded break in the spacer sequence. (C) Dual RNA-guided CRISPR-Cas9 nickase system. In this system, two sgRNAs are expressed that each guide a mutant version of Cas9 (Cas9-D10A) (that only nicks one strand of the DNA rather than making a double-stranded cut) to two different sequences that flank the target region. The two Cas9 nickases bind to opposite strands of the DNA nicking both DNA strands flanking the target region. This introduces a site-specific double-stranded break that is then repaired by NHEJ.

Mentions: The low but still measurable frequency of off-target mutations in the single Cas systems discussed above has been improved to essentially undetectable levels by the development of dual cas platforms (Table 1). To increase the specificity of RNA-guided nucleases, the dimerization-dependent Fok1 nuclease domain was fused to a catalytically inactive Cas9 (dCas9) protein [46]. In this system, sequence-specific DNA cleavage only occurs upon dimerization of two Fok1 nuclease domains from two different RNA-guided Fok1 nucleases (RFNs) that are bound in close proximity to two unique target sites (called half-sites) (Figure 2A, B). To be fully effective, the half-sites must be 14–17 bp apart and the entire target sequence must be flanked on the 5′ end by the sequence 5′-CCN and flanked on the 3′ end by the sequence NGG-3′. This system requires the use of two sgRNAs (one for each half-site). As the authors note, a full 44 bp RFN target site would almost always be unique in the genome unless located in a duplicated area of the genome. However, to assess the potential of off-targeting by RFNs, all sites in the genome that most closely matched the target regions of 3 RFNs were identified. Deep sequencing analysis of these areas following RFN-directed target mutagenesis detected no mutations above background (Table 1). This data suggests that RFN technology offers extraordinary precision. On-target mutation frequencies induced by RFNs at 12 different target sites in 9 different human genes ranged between 3 and 40% (Table 1). Some important cost advantages to this platform are that the plasmids to express the Cas9/Fok1 fusion proteins and the sgRNAs are inexpensive to purchase and the software to locate suitable target sequences against a gene of interest is publicly available. However, if one is generating an extensive library of RFNs against hundreds or thousands of genes, then the additional costs of generating two sgRNAs per target for the RFN technology may be a concern [46].Figure 2


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

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

Dual CRISPR-Cas technologies that increase nuclease specificity. (A, B) Dimeric CRISPR RNA-guided Fok1 nuclease target sequences consist of two 20 nt half-sites flanked by a protospacer adjacent motif (PAM) sequence in the form of 5′-NGG that are separated by a 14–17 nt spacer sequence. Each half-site is bound by a Cas-9/Fok1 fusion protein. Once bound, the Fok1 domains of two different Cas-9/Fok1 fusion proteins dimerize and introduce a double-stranded break in the spacer sequence. (C) Dual RNA-guided CRISPR-Cas9 nickase system. In this system, two sgRNAs are expressed that each guide a mutant version of Cas9 (Cas9-D10A) (that only nicks one strand of the DNA rather than making a double-stranded cut) to two different sequences that flank the target region. The two Cas9 nickases bind to opposite strands of the DNA nicking both DNA strands flanking the target region. This introduces a site-specific double-stranded break that is then repaired by NHEJ.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Dual CRISPR-Cas technologies that increase nuclease specificity. (A, B) Dimeric CRISPR RNA-guided Fok1 nuclease target sequences consist of two 20 nt half-sites flanked by a protospacer adjacent motif (PAM) sequence in the form of 5′-NGG that are separated by a 14–17 nt spacer sequence. Each half-site is bound by a Cas-9/Fok1 fusion protein. Once bound, the Fok1 domains of two different Cas-9/Fok1 fusion proteins dimerize and introduce a double-stranded break in the spacer sequence. (C) Dual RNA-guided CRISPR-Cas9 nickase system. In this system, two sgRNAs are expressed that each guide a mutant version of Cas9 (Cas9-D10A) (that only nicks one strand of the DNA rather than making a double-stranded cut) to two different sequences that flank the target region. The two Cas9 nickases bind to opposite strands of the DNA nicking both DNA strands flanking the target region. This introduces a site-specific double-stranded break that is then repaired by NHEJ.
Mentions: The low but still measurable frequency of off-target mutations in the single Cas systems discussed above has been improved to essentially undetectable levels by the development of dual cas platforms (Table 1). To increase the specificity of RNA-guided nucleases, the dimerization-dependent Fok1 nuclease domain was fused to a catalytically inactive Cas9 (dCas9) protein [46]. In this system, sequence-specific DNA cleavage only occurs upon dimerization of two Fok1 nuclease domains from two different RNA-guided Fok1 nucleases (RFNs) that are bound in close proximity to two unique target sites (called half-sites) (Figure 2A, B). To be fully effective, the half-sites must be 14–17 bp apart and the entire target sequence must be flanked on the 5′ end by the sequence 5′-CCN and flanked on the 3′ end by the sequence NGG-3′. This system requires the use of two sgRNAs (one for each half-site). As the authors note, a full 44 bp RFN target site would almost always be unique in the genome unless located in a duplicated area of the genome. However, to assess the potential of off-targeting by RFNs, all sites in the genome that most closely matched the target regions of 3 RFNs were identified. Deep sequencing analysis of these areas following RFN-directed target mutagenesis detected no mutations above background (Table 1). This data suggests that RFN technology offers extraordinary precision. On-target mutation frequencies induced by RFNs at 12 different target sites in 9 different human genes ranged between 3 and 40% (Table 1). Some important cost advantages to this platform are that the plasmids to express the Cas9/Fok1 fusion proteins and the sgRNAs are inexpensive to purchase and the software to locate suitable target sequences against a gene of interest is publicly available. However, if one is generating an extensive library of RFNs against hundreds or thousands of genes, then the additional costs of generating two sgRNAs per target for the RFN technology may be a concern [46].Figure 2

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