CRISPR
Encyclopedia
CRISPRs are loci
containing multiple short direct repeats that are found in the genomes of approximately 40% of bacteria
and 90% of archaea
. CRISPR functions as a prokaryotic immune system, in that it confers resistance to exogenous genetic elements such as plasmids and phages. The CRISPR system provides a form of acquired immunity. Short segments of foreign DNA, called spacers, are incorporated into the genome between CRISPR repeats, and serve as a 'memory' of past exposures. CRISPR spacers are then used to recognize and silence exogenous genetic elements in a manner analogous to RNAi
in eukaryotic organisms.
. In 2000, similar clustered repeats were identified in the genomes of additional bacteria and archaea, and were termed Short Regularly Spaced Repeats (SRSR). SRSR were renamed CRISPR in 2002. A set of genes, some encoding putative nuclease
or helicase
proteins, were found to be associated with CRISPR repeats (the cas, or CRISPR-associated, genes).
, implying the formation of a secondary structure
such as a hairpin
, but are not truly palindromic. CRISPR repeats are separated by spacers of similar length. Some CRISPR spacer sequences have identity to sequences from plasmids and phage, although some spacers have identity to the prokaryote's own genome (self-targeting spacers). New spacers can be added rapidly in response to phage infection.
families have been described. Of these protein families, Cas1 appears to be ubiquitous among different CRISPR/Cas systems. Particular combinations of cas genes and repeat structures have been used to define 8 CRISPR subtypes (Ecoli, Ypest, Nmeni, Dvulg, Tneap, Hmari, Apern, and Mtube), some of which are associated with an additional gene module encoding repeat-associated mysterious proteins (RAMPs). More than one CRISPR subtype may occur in a single genome. The sporadic distribution of the CRISPR/Cas subtypes suggests that the system is subject to horizontal gene transfer
during microbial evolution
.
Through the CRISPR-Cas mechanism bacteria can acquire immunity
against certain phages and thus halt further transmission of targeted phages. For this reason, some researchers have proposed that the CRISPR-Cas system is a Lamarckian
inheritance mechanism. Others investigated the coevolution of host and viral genomes by analysis of CRISPR sequences.
There are several proposals for CRISPR-derived biotechnology:
Locus (genetics)
In the fields of genetics and genetic computation, a locus is the specific location of a gene or DNA sequence on a chromosome. A variant of the DNA sequence at a given locus is called an allele. The ordered list of loci known for a particular genome is called a genetic map...
containing multiple short direct repeats that are found in the genomes of approximately 40% of bacteria
Bacteria
Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...
and 90% of archaea
Archaea
The Archaea are a group of single-celled microorganisms. A single individual or species from this domain is called an archaeon...
. CRISPR functions as a prokaryotic immune system, in that it confers resistance to exogenous genetic elements such as plasmids and phages. The CRISPR system provides a form of acquired immunity. Short segments of foreign DNA, called spacers, are incorporated into the genome between CRISPR repeats, and serve as a 'memory' of past exposures. CRISPR spacers are then used to recognize and silence exogenous genetic elements in a manner analogous to RNAi
RNAI
RNAI is a non-coding RNA that is an antisense repressor of the replication of some E. coli plasmids, including ColE1. Plasmid replication is usually initiated by RNAII, which acts as a primer by binding to its template DNA. The complementary RNAI binds RNAII prohibiting it from its initiation role...
in eukaryotic organisms.
Discovery of CRISPR
The clustered genomic repeats that are today known as CRISPR were first described in 1987 for the bacterium Escherichia coliEscherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
. In 2000, similar clustered repeats were identified in the genomes of additional bacteria and archaea, and were termed Short Regularly Spaced Repeats (SRSR). SRSR were renamed CRISPR in 2002. A set of genes, some encoding putative nuclease
Nuclease
A nuclease is an enzyme capable of cleaving the phosphodiester bonds between the nucleotide subunits of nucleic acids. Older publications may use terms such as "polynucleotidase" or "nucleodepolymerase"....
or helicase
Helicase
Helicases are a class of enzymes vital to all living organisms. They are motor proteins that move directionally along a nucleic acid phosphodiester backbone, separating two annealed nucleic acid strands using energy derived from ATP hydrolysis.-Function:Many cellular processes Helicases are a...
proteins, were found to be associated with CRISPR repeats (the cas, or CRISPR-associated, genes).
CRISPR repeats and spacers
CRISPR repeats range in size from 24 to 48 base pairs. They usually show some dyad symmetryDyad symmetry
In genetics, dyad symmetry refers to two areas of a DNA strand whose base pair sequences are inverted repeats of each other. They are often described as palindromes....
, implying the formation of a secondary structure
Nucleic acid secondary structure
The secondary structure of a nucleic acid molecule refers to the basepairing interactions within a single molecule or set of interacting molecules, and can be represented as a list of bases which are paired in a nucleic acid molecule....
such as a hairpin
Stem-loop
Stem-loop intramolecular base pairing is a pattern that can occur in single-stranded DNA or, more commonly, in RNA. The structure is also known as a hairpin or hairpin loop. It occurs when two regions of the same strand, usually complementary in nucleotide sequence when read in opposite directions,...
, but are not truly palindromic. CRISPR repeats are separated by spacers of similar length. Some CRISPR spacer sequences have identity to sequences from plasmids and phage, although some spacers have identity to the prokaryote's own genome (self-targeting spacers). New spacers can be added rapidly in response to phage infection.
cas genes and CRISPR subtypes
The CRISPR-associated (cas) genes are often associated with CRISPR repeat-spacer arrays. More than forty different Cas proteinProtein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
families have been described. Of these protein families, Cas1 appears to be ubiquitous among different CRISPR/Cas systems. Particular combinations of cas genes and repeat structures have been used to define 8 CRISPR subtypes (Ecoli, Ypest, Nmeni, Dvulg, Tneap, Hmari, Apern, and Mtube), some of which are associated with an additional gene module encoding repeat-associated mysterious proteins (RAMPs). More than one CRISPR subtype may occur in a single genome. The sporadic distribution of the CRISPR/Cas subtypes suggests that the system is subject to horizontal gene transfer
Horizontal gene transfer
Horizontal gene transfer , also lateral gene transfer , is any process in which an organism incorporates genetic material from another organism without being the offspring of that organism...
during microbial evolution
Evolution
Evolution is any change across successive generations in the heritable characteristics of biological populations. Evolutionary processes give rise to diversity at every level of biological organisation, including species, individual organisms and molecules such as DNA and proteins.Life on Earth...
.
CRISPR mechanism
Exogenous DNA is apparently processed by proteins encoded by some of the CRISPR-associated (cas) genes into small elements (of ~30bp in length), which are then somehow inserted into the CRISPR locus near the leader sequence. RNAs from the CRISPR loci are constituitively expressed and are processed by Cas proteins to small RNAs composed of individual exogenously-derived sequence elements with some flanking repeat sequence. The RNAs guide other Cas proteins to silence exogenous genetic elements at the RNA or DNA level. There is evidence for functional diversity among the different CRISPR subtypes. The Cse (Cas subtype Ecoli) proteins (called CasA-E in E. coli) form a functional complex, Cascade, that processes CRISPR RNA transcripts into spacer-repeat units that are retained by Cascade. In other prokaryotes, Cas6 processes the CRISPR transcripts. Interestingly, CRISPR-based phage inactivation in E. coli requires Cascade and Cas3, but not Cas1 and Cas2. The Cmr (Cas RAMP module) proteins found in Pyrococcus furiosus and other prokaryotes form a functional complex with small CRISPR RNAs that recognizes and cleaves complementary target RNAs.Evolutionary significance and possible applications
A bioinformatic study has shown that the CRISPRs are evolutionarily conserved and cluster into related types. Many show signs of a conserved secondary structure.Through the CRISPR-Cas mechanism bacteria can acquire immunity
Immunity (medical)
Immunity is a biological term that describes a state of having sufficient biological defenses to avoid infection, disease, or other unwanted biological invasion. Immunity involves both specific and non-specific components. The non-specific components act either as barriers or as eliminators of wide...
against certain phages and thus halt further transmission of targeted phages. For this reason, some researchers have proposed that the CRISPR-Cas system is a Lamarckian
Lamarckism
Lamarckism is the idea that an organism can pass on characteristics that it acquired during its lifetime to its offspring . It is named after the French biologist Jean-Baptiste Lamarck , who incorporated the action of soft inheritance into his evolutionary theories...
inheritance mechanism. Others investigated the coevolution of host and viral genomes by analysis of CRISPR sequences.
There are several proposals for CRISPR-derived biotechnology:
- Artificial immunization against phage by introduction of engineered CRISPR loci in industrially important bacteria, including those used in food production and large-scale fermentations.
- KnockdownKnockdownKnockdown may refer to:*"Knockdown" , a song by Alesha Dixon* Knockdown, in full-contact combat sports, when a fighter is down or vulnerable, often preliminary to a knockout...
of endogenous genes by transformation with a plasmidPlasmidIn microbiology and genetics, a plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double-stranded and, in many cases, circular...
which contains a CRISPR area with a spacer, which inhibits a target gene. - Discrimination of different bacterial strains by comparison of CRISPR spacer sequences (spoligotyping).
External links
- http://crispr.u-psud.fr/Server/CRISPRfinder.php
- http://crispr.u-psud.fr/crispr/
- http://crispr.u-psud.fr/CRISPRcompar/
- Rfam page for the CRISPR entries