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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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Related in: MedlinePlus

The CRISPR-Cas9 bacterial immune system and design of a CRISPR-Cas9 target sequence. (A) The CRISPR-Cas system acts as an adaptive immune system in bacteria and archaea. Clustered regularly interspaced short palindromic repeats (CRISPR) regions are stretches of repetitive genomic bacterial or archaea DNA interspersed by segments of foreign DNA sequences captured from bacterial phages and plasmids. A cluster of Cas (CRISPR associated) genes are located near the CRISPR region. The Cas9 gene, which is unique to type II CRISPR systems, codes for an RNA-guided endonuclease. Following foreign DNA infection in type II CRISPR systems, the CRISPR region is transcribed as a single RNA transcript called a pre-crRNA, and in type II systems, the pre-crRNAs are bound by tracrRNAs, processed into individual crRNA:tracrRNA duplexes by RNase III and form a complex with Cas9. The crRNA sequences are complementary to the foreign DNA and direct the Cas9 nuclease to form a complex with the foreign DNA and introduce a double-stranded break. (B) CRISPR-Cas9 target sequences are 20-nt long and are flanked by a protospacer adjacent motif (PAM) sequence in the form of 5′-NGG.
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Fig1: The CRISPR-Cas9 bacterial immune system and design of a CRISPR-Cas9 target sequence. (A) The CRISPR-Cas system acts as an adaptive immune system in bacteria and archaea. Clustered regularly interspaced short palindromic repeats (CRISPR) regions are stretches of repetitive genomic bacterial or archaea DNA interspersed by segments of foreign DNA sequences captured from bacterial phages and plasmids. A cluster of Cas (CRISPR associated) genes are located near the CRISPR region. The Cas9 gene, which is unique to type II CRISPR systems, codes for an RNA-guided endonuclease. Following foreign DNA infection in type II CRISPR systems, the CRISPR region is transcribed as a single RNA transcript called a pre-crRNA, and in type II systems, the pre-crRNAs are bound by tracrRNAs, processed into individual crRNA:tracrRNA duplexes by RNase III and form a complex with Cas9. The crRNA sequences are complementary to the foreign DNA and direct the Cas9 nuclease to form a complex with the foreign DNA and introduce a double-stranded break. (B) CRISPR-Cas9 target sequences are 20-nt long and are flanked by a protospacer adjacent motif (PAM) sequence in the form of 5′-NGG.

Mentions: Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems are adaptive immune systems used by many bacteria and archaea to fight off foreign DNA in the form of bacterial phages and/or plasmids [1-5]. Although multiple CRISPR-Cas types (I, II, and III) and subtypes (I-A to I-F) have been identified in bacteria and archaea, we pay particular attention to the specifics of the type II since type II has been engineered and adapted for use in eukaryotic systems, which is the focus of this review. Generally, the CRISPR-Cas system works through RNA-directed endonuclease cleavage of the invading genomic sequence. The invading sequence is captured and inserted directly into the genome of the host organism between CRISPR regions (Figure 1A) [6-8]. Following foreign DNA infection, the sequences within the CRISPR regions are transcribed as a single RNA transcript called a precursor CRISPR RNA (pre-crRNA). In the CRISPR-Cas9 system, the pre-crRNAs are bound by additional RNAs termed transactivating CRISPR RNAs (tracrRNAs) [9-12]. Once bound, the pre-crRNAs are processed into individual crRNA:tracrRNA duplexes by RNase III and together form a complex with an endonuclease [9-12]. The endonuclease Cas9 that is encoded from a region of the host genome adjacent to the CRISPR region is directed to the invading DNA in a sequence-dependent manner via the crRNA. Once bound to the foreign DNA, Cas9 introduces a double-stranded break in the foreign DNA [11-13].Figure 1


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

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

The CRISPR-Cas9 bacterial immune system and design of a CRISPR-Cas9 target sequence. (A) The CRISPR-Cas system acts as an adaptive immune system in bacteria and archaea. Clustered regularly interspaced short palindromic repeats (CRISPR) regions are stretches of repetitive genomic bacterial or archaea DNA interspersed by segments of foreign DNA sequences captured from bacterial phages and plasmids. A cluster of Cas (CRISPR associated) genes are located near the CRISPR region. The Cas9 gene, which is unique to type II CRISPR systems, codes for an RNA-guided endonuclease. Following foreign DNA infection in type II CRISPR systems, the CRISPR region is transcribed as a single RNA transcript called a pre-crRNA, and in type II systems, the pre-crRNAs are bound by tracrRNAs, processed into individual crRNA:tracrRNA duplexes by RNase III and form a complex with Cas9. The crRNA sequences are complementary to the foreign DNA and direct the Cas9 nuclease to form a complex with the foreign DNA and introduce a double-stranded break. (B) CRISPR-Cas9 target sequences are 20-nt long and are flanked by a protospacer adjacent motif (PAM) sequence in the form of 5′-NGG.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: The CRISPR-Cas9 bacterial immune system and design of a CRISPR-Cas9 target sequence. (A) The CRISPR-Cas system acts as an adaptive immune system in bacteria and archaea. Clustered regularly interspaced short palindromic repeats (CRISPR) regions are stretches of repetitive genomic bacterial or archaea DNA interspersed by segments of foreign DNA sequences captured from bacterial phages and plasmids. A cluster of Cas (CRISPR associated) genes are located near the CRISPR region. The Cas9 gene, which is unique to type II CRISPR systems, codes for an RNA-guided endonuclease. Following foreign DNA infection in type II CRISPR systems, the CRISPR region is transcribed as a single RNA transcript called a pre-crRNA, and in type II systems, the pre-crRNAs are bound by tracrRNAs, processed into individual crRNA:tracrRNA duplexes by RNase III and form a complex with Cas9. The crRNA sequences are complementary to the foreign DNA and direct the Cas9 nuclease to form a complex with the foreign DNA and introduce a double-stranded break. (B) CRISPR-Cas9 target sequences are 20-nt long and are flanked by a protospacer adjacent motif (PAM) sequence in the form of 5′-NGG.
Mentions: Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems are adaptive immune systems used by many bacteria and archaea to fight off foreign DNA in the form of bacterial phages and/or plasmids [1-5]. Although multiple CRISPR-Cas types (I, II, and III) and subtypes (I-A to I-F) have been identified in bacteria and archaea, we pay particular attention to the specifics of the type II since type II has been engineered and adapted for use in eukaryotic systems, which is the focus of this review. Generally, the CRISPR-Cas system works through RNA-directed endonuclease cleavage of the invading genomic sequence. The invading sequence is captured and inserted directly into the genome of the host organism between CRISPR regions (Figure 1A) [6-8]. Following foreign DNA infection, the sequences within the CRISPR regions are transcribed as a single RNA transcript called a precursor CRISPR RNA (pre-crRNA). In the CRISPR-Cas9 system, the pre-crRNAs are bound by additional RNAs termed transactivating CRISPR RNAs (tracrRNAs) [9-12]. Once bound, the pre-crRNAs are processed into individual crRNA:tracrRNA duplexes by RNase III and together form a complex with an endonuclease [9-12]. The endonuclease Cas9 that is encoded from a region of the host genome adjacent to the CRISPR region is directed to the invading DNA in a sequence-dependent manner via the crRNA. Once bound to the foreign DNA, Cas9 introduces a double-stranded break in the foreign DNA [11-13].Figure 1

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
Related in: MedlinePlus