CRISPR-Cas9: Another Tool for Editing Genomes

Some bacteria have a system for recognizing an invading virus and destroying it. The system is called CRISPR (for clustered regularly interspaced short palindromic repeats) [Link to a discussion of palindromes.] It is described on a separate page — Link to it.

Like restriction enzymes (also discovered in bacteria), CRISPR provides a way of targeting a particular DNA sequence. The CRISPR gene encodes an RNA (crRNA) that contains a length of 23 nucleotides that will bind by Watson-Crick base-pairing to the complementary sequence on a strand of DNA.

The CRISPR RNA molecule also binds to an endonuclease called Cas9 that cuts both strands of the DNA within the target sequence.

Although found in bacteria, the CRISPR-Cas9 system can be introduced into eukaryotic cells where it enables specific genes to be altered.

Example:

The CRISPR-Cas9 genes can be introduced into a eukaryotic cell on a plasmid. Expression of the crRNA and the Cas9 protein cuts both strands of the target DNA sequence. When the host DNA repair enzymes repair the double-stranded break (DSB) by NonHomologous End-Joining (NHEJ), this error-prone process is apt to introduce insertions or deletions (indels) into the DNA sequence. Often such indels will create a frameshift thus inactivating the gene.

Another Example:

One can introduce a plasmid containing the CRISPR-Cas9 genes AND a gene that one hopes to incorporate into the host cell's genome. If the double-stranded break is repaired by homologous recombination (homology-directed repair), the introduced gene can be inserted into the site.

Genome editing with the CRISPR-Cas9 system has worked successfully in a variety of animals, plants and microbes. It has even been able to correct a mutant gene in the cells of human embryos (which were not allowed to go on to develop in vivo).

Gene Drive: A Third Example

Researchers at the San Diego campus of the University of California have taken the process one remarkable step further. Their plasmid contains:

• a CRISPR gene encoding a crRNA that binds to a gene (y) involved in Drosophila pigmentation.
• a Cas9 gene that cuts the y gene. Disruption of this recessive X-linked gene produces yellow flies when the normal allele is absent.
• flanking DNA sequences that enable the CRISPR-Cas9 genes to be physically incorporated into the y locus of a host X chromosome.

Expression of these genes produces more y-targeting crRNA and more Cas9. The result: the y locus on the second X chromosomes (in females) becomes inactivated as well. A heterozygous mutation has been converted into a homozygous mutation. The process is called gene drive or the mutagenic chain reaction.

One demonstration:

The females produced after injecting Drosophila embryos with the plasmid were mated with normal ("wild-type") males.

Normal Mendelian inheritance would have predicted that only the male offspring would have been yellow. The females would have inherited a normal y allele (black) from their fathers.

In fact, almost all (97%) of the flies, both sexes, were yellow. The mutant allele (red) inherited from their mothers converted the normal allele inherited from their fathers into the mutant form.

A second demonstration:

When mutant males were mated with wild-type females, Mendelian inheritance would predict that all the offspring would be normal.

In fact, while all the males were normal, all the females were yellow. The mutant allele inherited from their fathers converted the normal allele inherited from their mothers into the mutant form.

Implications of Gene Drive Technology

When a new mutant gene appears in a population, its frequency in that population may slowly increase (or decrease) if its phenotype confers a selective advantage (or disadvantage) on those carrying it (Natural Selection).

Even if there is there is no effect on survival, its frequency may very slowly increase or decrease by genetic drift.

But if the new allele has been engineered to trigger a mutagenic chain reaction, that allele will quickly sweep through the population turning all offspring into homozygotes expressing the allele.

Gene drive technology could have many beneficial effects.

For example, introducing a mutagenic chain reaction of a gene that blocks the ability of mosquitoes to transmit malaria could theoretically quickly end disease transmission.

However, like all technologies, gene drive poses risks as well. The ability to have an engineered gene sweep through a natural population raises the spectre of unexpected harmful consequences to entire ecosystems. For this reason, the workers in San Diego took elaborate precautions to be certain that their flies did not escape into the wild.