I-CreI

I-CreI has evolved to cut a 22-nucleotide sequence of DNA that occurs in alleles 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.^[3] 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.

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 genome. 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.^[5] In 1997, the structure of the I-CreI protein was determined,^[6] 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.^[4] Mutant forms of the protein have since been created that exhibit altered homing site specificity.^[7][8][9] A genetic system in Saccharomyces cerevisiae has also been created, yielding additional I-CreI mutants with modified homing site specificities.^[10][11]

I-CreI has already been used successfully to induce homologous recombination in Drosophila melanogaster, an extremely popular eukaryotic model organism.^[12] 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.