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Application of CRISPR/Cas9 for biomedical discoveries.

Riordan SM, Heruth DP, Zhang LQ, Ye SQ - Cell Biosci (2015)

Bottom Line: The CRISPR/Cas9 system is much easier to customize and optimize because the site selection for DNA cleavage is guided by a short sequence of RNA rather than an engineered protein as in the systems of zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and meganucleases.In this review, we present a brief history and development of the CRISPR system and focus on the application of this genome editing technology for biomedical discoveries.We then present concise concluding remarks and future directions for this fast moving field.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, The Children's Mercy Hospital, Kansas City, MO USA.

ABSTRACT
The Clustered Regions of Interspersed Palindromic Repeats-Cas9 (CRISPR/Cas9), a viral defense system found in bacteria and archaea, has emerged as a tour de force genome editing tool. The CRISPR/Cas9 system is much easier to customize and optimize because the site selection for DNA cleavage is guided by a short sequence of RNA rather than an engineered protein as in the systems of zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and meganucleases. Although it still suffers from some off-target effects, the CRISPR/Cas9 system has been broadly and successfully applied for biomedical discoveries in a number of areas. In this review, we present a brief history and development of the CRISPR system and focus on the application of this genome editing technology for biomedical discoveries. We then present concise concluding remarks and future directions for this fast moving field.

No MeSH data available.


Related in: MedlinePlus

Mechanism of Cas9 action. a The three essential components necessary for CRISPR/Cas activity, using spCas9 as an example, are Cas9, crRNA and tracrRNA. The introduction of the linker region to combine the crRNA and tracrRNA into a single guide RNA (sgRNA) improves overall targeting efficiency. b Recognition of the protospacer region by the sgRNA in conjunction with Cas9 recognition of the appropriate PAM sequence initiates cleavage. c Cleaved DNA fragments. d Mutated forms of Cas9 can contain a single active domain to act as a Cas9n “nickase” where only one strand of the DNA is cleaved. e Alternatively both catalytic domains can be mutated to form a dCas9 “dead” nuclease that can then be tethered to effector molecules to target repression or activation, KRAB and VP64 respectively, for example. Images adapted from motifolio.com
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Fig2: Mechanism of Cas9 action. a The three essential components necessary for CRISPR/Cas activity, using spCas9 as an example, are Cas9, crRNA and tracrRNA. The introduction of the linker region to combine the crRNA and tracrRNA into a single guide RNA (sgRNA) improves overall targeting efficiency. b Recognition of the protospacer region by the sgRNA in conjunction with Cas9 recognition of the appropriate PAM sequence initiates cleavage. c Cleaved DNA fragments. d Mutated forms of Cas9 can contain a single active domain to act as a Cas9n “nickase” where only one strand of the DNA is cleaved. e Alternatively both catalytic domains can be mutated to form a dCas9 “dead” nuclease that can then be tethered to effector molecules to target repression or activation, KRAB and VP64 respectively, for example. Images adapted from motifolio.com

Mentions: The CRISPR system loci is defined by the presence of the 20 – 50 base pair repeats separated by the unique spacer sequences acquired from invading viral and plasmid DNA [29]. In the type II CRISPR system, spacer acquisition depends on Cas1, Cas2, Csn2, and Cas9. While Csn2 and Cas9 are unique to the type II system, Cas1 and Cas2 seem to be common to all three CRISRP types [15, 18]. For the type II system there is evidence that Cas9 is integral in the initial recognition of the potential protospacer + PAM sequence in the invading DNA [15]. This dual role for Cas9 is a logical mechanism to ensure that the protospacers selected will be present next to the required PAM sequence so that Cas9 will be able to recognize it for future targeting and cleavage when challenged. Following transcription, RNAse III and Cas9 process the pre-crRNA transcript into the mature crRNA forms [30]. In addition to processing the pre-crRNA transcript, Cas9 also functions as the sole endonuclease necessary for the introduction of DSBs in the Type II CRISPR system. Cas9 is guided to its target sequence after forming a complex with the crRNA and a tracrRNA. TracrRNA forms a duplex with the crRNA and is essential for the proper Cas9:crRNA interaction and recognition of the target DNA sequence (Fig. 2) [30].Fig. 2


Application of CRISPR/Cas9 for biomedical discoveries.

Riordan SM, Heruth DP, Zhang LQ, Ye SQ - Cell Biosci (2015)

Mechanism of Cas9 action. a The three essential components necessary for CRISPR/Cas activity, using spCas9 as an example, are Cas9, crRNA and tracrRNA. The introduction of the linker region to combine the crRNA and tracrRNA into a single guide RNA (sgRNA) improves overall targeting efficiency. b Recognition of the protospacer region by the sgRNA in conjunction with Cas9 recognition of the appropriate PAM sequence initiates cleavage. c Cleaved DNA fragments. d Mutated forms of Cas9 can contain a single active domain to act as a Cas9n “nickase” where only one strand of the DNA is cleaved. e Alternatively both catalytic domains can be mutated to form a dCas9 “dead” nuclease that can then be tethered to effector molecules to target repression or activation, KRAB and VP64 respectively, for example. Images adapted from motifolio.com
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4487574&req=5

Fig2: Mechanism of Cas9 action. a The three essential components necessary for CRISPR/Cas activity, using spCas9 as an example, are Cas9, crRNA and tracrRNA. The introduction of the linker region to combine the crRNA and tracrRNA into a single guide RNA (sgRNA) improves overall targeting efficiency. b Recognition of the protospacer region by the sgRNA in conjunction with Cas9 recognition of the appropriate PAM sequence initiates cleavage. c Cleaved DNA fragments. d Mutated forms of Cas9 can contain a single active domain to act as a Cas9n “nickase” where only one strand of the DNA is cleaved. e Alternatively both catalytic domains can be mutated to form a dCas9 “dead” nuclease that can then be tethered to effector molecules to target repression or activation, KRAB and VP64 respectively, for example. Images adapted from motifolio.com
Mentions: The CRISPR system loci is defined by the presence of the 20 – 50 base pair repeats separated by the unique spacer sequences acquired from invading viral and plasmid DNA [29]. In the type II CRISPR system, spacer acquisition depends on Cas1, Cas2, Csn2, and Cas9. While Csn2 and Cas9 are unique to the type II system, Cas1 and Cas2 seem to be common to all three CRISRP types [15, 18]. For the type II system there is evidence that Cas9 is integral in the initial recognition of the potential protospacer + PAM sequence in the invading DNA [15]. This dual role for Cas9 is a logical mechanism to ensure that the protospacers selected will be present next to the required PAM sequence so that Cas9 will be able to recognize it for future targeting and cleavage when challenged. Following transcription, RNAse III and Cas9 process the pre-crRNA transcript into the mature crRNA forms [30]. In addition to processing the pre-crRNA transcript, Cas9 also functions as the sole endonuclease necessary for the introduction of DSBs in the Type II CRISPR system. Cas9 is guided to its target sequence after forming a complex with the crRNA and a tracrRNA. TracrRNA forms a duplex with the crRNA and is essential for the proper Cas9:crRNA interaction and recognition of the target DNA sequence (Fig. 2) [30].Fig. 2

Bottom Line: The CRISPR/Cas9 system is much easier to customize and optimize because the site selection for DNA cleavage is guided by a short sequence of RNA rather than an engineered protein as in the systems of zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and meganucleases.In this review, we present a brief history and development of the CRISPR system and focus on the application of this genome editing technology for biomedical discoveries.We then present concise concluding remarks and future directions for this fast moving field.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, The Children's Mercy Hospital, Kansas City, MO USA.

ABSTRACT
The Clustered Regions of Interspersed Palindromic Repeats-Cas9 (CRISPR/Cas9), a viral defense system found in bacteria and archaea, has emerged as a tour de force genome editing tool. The CRISPR/Cas9 system is much easier to customize and optimize because the site selection for DNA cleavage is guided by a short sequence of RNA rather than an engineered protein as in the systems of zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and meganucleases. Although it still suffers from some off-target effects, the CRISPR/Cas9 system has been broadly and successfully applied for biomedical discoveries in a number of areas. In this review, we present a brief history and development of the CRISPR system and focus on the application of this genome editing technology for biomedical discoveries. We then present concise concluding remarks and future directions for this fast moving field.

No MeSH data available.


Related in: MedlinePlus