CRISPR

CRISPR ( Clustered Regularly Spaced Inter Short Palindromic Repeats ) are sections of repetitive DNA ( repeats) that occur in the genomes of many bacteria and archaea. They serve a mechanism of resistance to the penetration of alien genotypes enforced by viruses or plasmids and are thus a part of the immune system equivalent of many prokaryotes.

Discovery and properties

The existence of the DNA segments, which are known as CRISPR today, was discovered in 1987 in the bacterium E. coli. In 2002 it was announced that similar structures in the genome of many different prokaryotes exist, and the name CRISPR was coined. In addition, a group was discovered by genes that were close in all organisms studied at the gene locus of the CRISPR and cas genes were therefore called ( CRISPR -associated ).

Today it is known that the genome of approximately 45% of the previously sequenced bacteria and 83% of Archaea comprises at least one CRISPR structure.

CRISPR have a length that varies from 23 to 47 bp. The individual sequences of repetitive basic motif alternating with spacers that have a length of 21-72 bp. While within a CRISPR structure, the repeating sequence is maintained, the sequence of the CRISPR varies greatly in different microorganisms. CRISPR repeats of the sequence is palindromic in general, resulting in a stable secondary structure of the corresponding RNA according.

The sequences of the spacer sections vary widely, both in structure of a CRISPR and in various prokaryotes. 2005, it was discovered that the spacer sequences are identical to the foreign DNA from bacteriophages and plasmids. This led to the hypothesis that the function of CRISPR is to defend the body against foreign DNA.

Pathogens of the species Francisella use the CRISPR - Cas system for immune evasion. With Neisseria meningitidis and Campylobacter jejuni, the system is a virulence factor to a hitherto unknown mechanism.

Immunity by CRISPR

2007 of Barrangou et al. shown that bacteria infected with phages integrate parts of the foreign DNA as a spacer in the CRISPR regions of their genome and thus can develop immunity to the phage. In addition, they showed that spacer sequences which are artificially inserted into the CRISPR regions of bacterial, these make them resistant to the related phages. If the spacer sequences cut out again, and the resistance is canceled. It was also shown that the cas genes play an essential role in phage defense: Disabling some cas genes ( cas1 ) prevents despite existing Spacer defense against phages. The activity of other cas genes ( CAS 7 ) is necessary for the integration of new spacers into the CRISPR sequence.

Mechanism

Despite great advances in recent years is the mechanism through which the CRISPR provides / cas system prokaryotes immunity, not yet well understood. It is assumed that the exogenous DNA is detected in the immunization process by Cas protein complex and integrated as a new spacers into the CRISPR regions. How to run these processes in detail, is still unknown.

The initial sequence of a CRISPR region acts as a promoter, and most likely to cause transcription of the entire CRISPR region in a long RNA molecule. This is then decomposed using a Cas complex ( Cascade ) into parts, each containing a spacer and on the edges of a part of the palindromic repeats. In conjunction with the Cas proteins, the individual RNA sequences can detect and disable foreign DNA or RNA and thus specifically give the prokaryotes immunization against those phage whose genome contains the spacer sequence in a likewise not yet precisely known mechanism. This mechanism has similarities to RNA interference in eukaryotes, but there are also significant differences, especially in the supporting protein machinery.

Evolutionary implications

Through the CRISPR - Cas mechanism bacteria can acquire immunity against certain phages, and further bequeath as acquired immunity, as they integrate a virus-specific spacer in their genome and thus to pass during replication. For this reason, the provocative thesis was expressed that it was the CRISPR - Cas system is the first truly Lamarckian inheritance mechanism. The molecular mechanism of CRISPR self, however, is caused by "classical" evolution.

Applications

There are several proposals CRISPR exploit biotechnology:

• Artificial immunization against phage by adding suitable spacers in industrially important bacteria, eg in milk or wine industry
• Knockdown of endogenous genes by transformation with a plasmid containing a CRISPR region includes a spacer which matches the be shut down gene
• Distinguish between different strains of bacteria by comparing the spacer regions ( spoligotyping )

The Cas - CRISPR system can be used for gene therapy. A green fluorescent protein can be used with a CAS mutant as a fusion protein for marker DNA sequences (including sequences such as telomeres repetitive ).