I-CreI
Encyclopedia
I-CreI is a homing endonuclease
whose gene
was first discovered in the chloroplast
genome of Chlamydomonas reinhardtii
, a species of unicellular green algae
. It is named for the facts that: it resides in an Intron; it was isolated from Clamydomonas reinhardtii; it was the first (I) such gene isolated from C. reinhardtii. Interestingly, its gene resides in a group I intron
in the 23S ribosomal RNA
gene of the C. reinhardtii chloroplast, and I-CreI is only expressed when its mRNA is spliced from the primary transcript
of the 23S gene. I-CreI enzyme
, which functions as a homodimer, recognizes a 22-nucleotide sequence of duplex DNA and cleaves one phosphodiester bond on each strand at specific positions. I-CreI is a member of the LAGLIDADG family of homing endonucleases, all of which have a conserved LAGLIDADG amino acid motif that contributes to their associative domains and active sites. When the I-CreI-containing intron encounters a 23S gene lacking the intron, I-CreI enzyme "homes" in on the "intron-minus" allele of 23S and effects its parent intron's insertion into the intron-minus allele. Introns with this behavior are called mobile introns. Because I-CreI provides for its own propagation while conferring no benefit on its host, it is an example of selfish DNA
.
in the 23S rRNA gene of the C. reinhardtii chloroplast genome. The 23S gene is an RNA gene, meaning that its transcript is not translated into protein. As RNA, it forms part of the large subunit of the ribosome
. An open reading frame coding for a 163-amino acid protein was found in this 23S intron, suggesting that a protein might facilitate the homing behavior of the mobile intron. Furthermore, the predicted protein had a LAGLIDADG motif, a conserved amino acid sequence that is present in other proteins coded for in group I mobile introns. A 1991 study established that the ORF codes for a DNA endonuclease, I-CreI, which selectively cuts a site corresponding to where the intron is spliced out of the 23S primary transcript. The study also showed that the intron was able to invade 23S alleles that did not already have it.
s of the 23S ribosomal RNA gene that lack the I-CreI-containing intron. When such an "intron-minus" allele is cut, pathways of double-strand break repair are activated in the cell. The cell uses as a template for repair the 23S allele that yielded the responsible I-CreI enzyme, thus replicating the I-CreI-containing intron. The resulting "intron-plus" allele no longer contains an intact homing site for the I-CreI enzyme, and is therefore not cleaved. Since this intron provides for its own replication without conferring any benefit on its host, I-CreI is a form of selfish DNA
.
. A four- or six-nucleotide sequence is expected to occur many, many times in a genome of millions or billions of nucleotides simply by chance, whereas a 22-nucleotide sequence might occur only once (109/46 vs. 109/422). This specificity of I-CreI cleavage makes I-CreI a promising tool for gene targeting
. If a person were to have a disease due to a defective allele
of some gene
, it would be helpful to be able to replace that allele with a functional one. If one could cause I-CreI to cut the DNA only in the defective allele while simultaneously providing a normal allele for the cell to use as a repair template, the patient's own homologous recombination
machinery could insert the desired allele in place of the dysfunctional one. The specificity of I-CreI also allows for the reduction of deleterious effects due to double-strand breaks outside of the gene of interest.
In order to use I-CreI as a tool in this fashion, it is necessary to make it recognize and cleave sequences of DNA different from its native homing site. An Escherichia coli
genetic system for studying the relationship between I-CreI structure and its homing site specificity was created in 1997. In 1997, the structure of the I-CreI protein was determined , and in 1998, its crystal structure bound to its native DNA homing site was solved, greatly aiding research in altering the homing site recognition of the protein. Mutant forms of the protein have since been created that exhibit altered homing site specificity. A genetic system in Saccharomyces cerevisiae
has also been created, yielding additional I-CreI mutants with modified homing site specificities.
I-CreI has already been used successfully to induce homologous recombination in Drosophila melanogaster
, an extremely popular eukaryotic
model organism
. It seems very likely that advances in molecular biological techniques and generation of a library of I-CreI-derived novel endonucleases will eventually allow for the targeting of many genes of etiological significance.
Homing endonuclease
The homing endonucleases are a type of restriction enzymes typically encoded by introns or inteins. They act on the cellular DNA of the cells that synthesize them, in the opposite alleles of the genes that encode them.- Origin and mechanism :...
whose gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
was first discovered in the chloroplast
Chloroplast
Chloroplasts are organelles found in plant cells and other eukaryotic organisms that conduct photosynthesis. Chloroplasts capture light energy to conserve free energy in the form of ATP and reduce NADP to NADPH through a complex set of processes called photosynthesis.Chloroplasts are green...
genome of Chlamydomonas reinhardtii
Chlamydomonas reinhardtii
Chlamydomonas reinhardtii is a single celled green alga about 10 micrometres in diameter that swims with two flagella. They have a cell wall made of hydroxyproline-rich glycoproteins, a large cup-shaped chloroplast, a large pyrenoid, and an "eyespot" that senses light.Although widely distributed...
, a species of unicellular green algae
Green algae
The green algae are the large group of algae from which the embryophytes emerged. As such, they form a paraphyletic group, although the group including both green algae and embryophytes is monophyletic...
. It is named for the facts that: it resides in an Intron; it was isolated from Clamydomonas reinhardtii; it was the first (I) such gene isolated from C. reinhardtii. Interestingly, its gene resides in a group I intron
Intron
An intron is any nucleotide sequence within a gene that is removed by RNA splicing to generate the final mature RNA product of a gene. The term intron refers to both the DNA sequence within a gene, and the corresponding sequence in RNA transcripts. Sequences that are joined together in the final...
in the 23S ribosomal RNA
Ribosomal RNA
Ribosomal ribonucleic acid is the RNA component of the ribosome, the enzyme that is the site of protein synthesis in all living cells. Ribosomal RNA provides a mechanism for decoding mRNA into amino acids and interacts with tRNAs during translation by providing peptidyl transferase activity...
gene of the C. reinhardtii chloroplast, and I-CreI is only expressed when its mRNA is spliced from the primary transcript
Transcription (genetics)
Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...
of the 23S gene. I-CreI enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
, which functions as a homodimer, recognizes a 22-nucleotide sequence of duplex DNA and cleaves one phosphodiester bond on each strand at specific positions. I-CreI is a member of the LAGLIDADG family of homing endonucleases, all of which have a conserved LAGLIDADG amino acid motif that contributes to their associative domains and active sites. When the I-CreI-containing intron encounters a 23S gene lacking the intron, I-CreI enzyme "homes" in on the "intron-minus" allele of 23S and effects its parent intron's insertion into the intron-minus allele. Introns with this behavior are called mobile introns. Because I-CreI provides for its own propagation while conferring no benefit on its host, it is an example of selfish DNA
Selfish DNA
Selfish DNA refers to those sequences of DNA which, in their purest form, have two distinct properties: the DNA sequence spreads by forming additional copies of itself within the genome; and it makes no specific contribution to the reproductive success of its host organism.This idea was sketched...
.
Discovery
I-CreI was first observed as an intervening sequenceIntron
An intron is any nucleotide sequence within a gene that is removed by RNA splicing to generate the final mature RNA product of a gene. The term intron refers to both the DNA sequence within a gene, and the corresponding sequence in RNA transcripts. Sequences that are joined together in the final...
in the 23S rRNA gene of the C. reinhardtii chloroplast genome. The 23S gene is an RNA gene, meaning that its transcript is not translated into protein. As RNA, it forms part of the large subunit of the ribosome
Ribosome
A ribosome is a component of cells that assembles the twenty specific amino acid molecules to form the particular protein molecule determined by the nucleotide sequence of an RNA molecule....
. An open reading frame coding for a 163-amino acid protein was found in this 23S intron, suggesting that a protein might facilitate the homing behavior of the mobile intron. Furthermore, the predicted protein had a LAGLIDADG motif, a conserved amino acid sequence that is present in other proteins coded for in group I mobile introns. A 1991 study established that the ORF codes for a DNA endonuclease, I-CreI, which selectively cuts a site corresponding to where the intron is spliced out of the 23S primary transcript. The study also showed that the intron was able to invade 23S alleles that did not already have it.
Mechanism of propagation
I-CreI has evolved to cut a 22-nucleotide sequence of DNA that occurs in alleleAllele
An allele is one of two or more forms of a gene or a genetic locus . "Allel" is an abbreviation of allelomorph. Sometimes, different alleles can result in different observable phenotypic traits, such as different pigmentation...
s of the 23S ribosomal RNA gene that lack the I-CreI-containing intron. When such an "intron-minus" allele is cut, pathways of double-strand break repair are activated in the cell. The cell uses as a template for repair the 23S allele that yielded the responsible I-CreI enzyme, thus replicating the I-CreI-containing intron. The resulting "intron-plus" allele no longer contains an intact homing site for the I-CreI enzyme, and is therefore not cleaved. Since this intron provides for its own replication without conferring any benefit on its host, I-CreI is a form of selfish DNA
Selfish DNA
Selfish DNA refers to those sequences of DNA which, in their purest form, have two distinct properties: the DNA sequence spreads by forming additional copies of itself within the genome; and it makes no specific contribution to the reproductive success of its host organism.This idea was sketched...
.
Structural studies and possible applications
Because I-CreI has evolved to cut such a long sequence of DNA, unlike restriction endonucleases that typically cut four- or six-nucleotide sequences, it is capable of cutting a single site within a very large genomeGenome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
. A four- or six-nucleotide sequence is expected to occur many, many times in a genome of millions or billions of nucleotides simply by chance, whereas a 22-nucleotide sequence might occur only once (109/46 vs. 109/422). This specificity of I-CreI cleavage makes I-CreI a promising tool for gene targeting
Gene therapy
Gene therapy is the insertion, alteration, or removal of genes within an individual's cells and biological tissues to treat disease. It is a technique for correcting defective genes that are responsible for disease development...
. If a person were to have a disease due to a defective allele
Allele
An allele is one of two or more forms of a gene or a genetic locus . "Allel" is an abbreviation of allelomorph. Sometimes, different alleles can result in different observable phenotypic traits, such as different pigmentation...
of some gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
, it would be helpful to be able to replace that allele with a functional one. If one could cause I-CreI to cut the DNA only in the defective allele while simultaneously providing a normal allele for the cell to use as a repair template, the patient's own homologous recombination
Homologous recombination
Homologous recombination is a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. It is most widely used by cells to accurately repair harmful breaks that occur on both strands of DNA, known as double-strand breaks...
machinery could insert the desired allele in place of the dysfunctional one. The specificity of I-CreI also allows for the reduction of deleterious effects due to double-strand breaks outside of the gene of interest.
In order to use I-CreI as a tool in this fashion, it is necessary to make it recognize and cleave sequences of DNA different from its native homing site. An Escherichia coli
Escherichia 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...
genetic system for studying the relationship between I-CreI structure and its homing site specificity was created in 1997. In 1997, the structure of the I-CreI protein was determined , and in 1998, its crystal structure bound to its native DNA homing site was solved, greatly aiding research in altering the homing site recognition of the protein. Mutant forms of the protein have since been created that exhibit altered homing site specificity. A genetic system in Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae is a species of yeast. It is perhaps the most useful yeast, having been instrumental to baking and brewing since ancient times. It is believed that it was originally isolated from the skin of grapes...
has also been created, yielding additional I-CreI mutants with modified homing site specificities.
I-CreI has already been used successfully to induce homologous recombination in Drosophila melanogaster
Drosophila melanogaster
Drosophila melanogaster is a species of Diptera, or the order of flies, in the family Drosophilidae. The species is known generally as the common fruit fly or vinegar fly. Starting from Charles W...
, an extremely popular eukaryotic
Eukaryote
A eukaryote is an organism whose cells contain complex structures enclosed within membranes. Eukaryotes may more formally be referred to as the taxon Eukarya or Eukaryota. The defining membrane-bound structure that sets eukaryotic cells apart from prokaryotic cells is the nucleus, or nuclear...
model organism
Model organism
A model organism is a non-human species that is extensively studied to understand particular biological phenomena, with the expectation that discoveries made in the organism model will provide insight into the workings of other organisms. Model organisms are in vivo models and are widely used to...
. It seems very likely that advances in molecular biological techniques and generation of a library of I-CreI-derived novel endonucleases will eventually allow for the targeting of many genes of etiological significance.