Correcting genetic defects with CRISPR–Cas9

Credit: PHOTOALTO

Two new studies show that the clustered regularly interspaced short palindromic repeats (CRISPR) method of genetic recombination can correct genetic defects in human adult stem cell organoids and in mouse zygotes.

The CRISPR–Cas9 (that is, CRISPR-associated) system uses specially designed RNAs that guide the Cas9 nuclease to the target DNA where it induces DNA breaks. These breaks are repaired either by non-homologous end-joining (NHEJ), which is an error-prone process that leads to other insertions or deletions, or by homology-directed repair (HDR), which requires a template but is less error prone.

Schwank and colleagues used this system to correct a mutation in the cystic fibrosis transmembrane conductance regulator gene (CFTR). They used intestinal organoids that were derived from adult stem cells from two cystic fibrosis patients who were homozygous for the same CFTR mutation. These organoids can be assayed for the presence of functional CFTR using a forskolin-induced swelling assay, in which organoids that lack the functional protein fail to swell. The authors transfected the organoids with a donor plasmid that contained both the wild-type CFTR sequence and the CRISPR–Cas9 guide RNA that targeted the mutant CFTR sequence.

Sequencing the transfected cells revealed successful repair and swelling in the forskolin assay that was comparable to wild-type organoids, which demonstrated functional repair of the mutant CFTR. The authors also searched for off-target mutations but found that these were rare: only one such mutation occurred in one of the organoids and was found within an intron, which makes phenotypic consequences less likely. This is especially exciting for using CRISPR–Cas9 in human patients, in whom off-target mutations are a safety concern.

In another study, Wu and colleagues used this system to correct a dominant mutation in the mouse crystallin, gamma C gene (Crygc). A 1 bp-deletion in this gene causes cataracts in both homozygous and heterozygote pups at weaning. The authors used heterozygous zygotes and injected them with guide RNAs that specifically targeted Cas9 to this mutation. The authors hypothesized that HDR following Cas9 nuclease-directed DNA cleavage could be used to correct this deletion using the wild-type allele in the heterozygotes as a template. Nearly half of the resultant pups had genetic alterations of the mutant allele, including insertions and deletions, as well as corrections. By contrast, none had alterations in the wild-type allele, which confirmed the specificity of the guide RNA. Moreover, all of the mice in which HDR-mediated repair had taken place carried corrected DNA and lacked cataracts, whereas only half of the mice in which NHEJ-mediated repair had occurred were cataract free.

The authors also determined whether an exogenous DNA oligonucleotide could be used as a template for HDR-mediated repair. To do this, they used an oligonucletide that encoded the same protein sequence as the wild-type allele but that also contained two synonymous mutations. This allowed the authors to determine whether the recombination template in HDR-mediated repair was the wild-type allele or the exogenous oligonucleotide. The majority of mice with corrected alleles used the exogenous oligonucleotide, which shows that, although exogenous oligonucleotides are not required, they could be templates for repairing genes. This is especially pertinent in homozygous mutants that lack a wild-type allele.

The authors also showed that the modified mice were fertile and that their offspring carried the repaired alleles. Similarly to the study by Schwank et al., off-target effects were rare: only 2 of 12 pups carried mutations at 1 of 10 potential off-target sites.

“these studies suggest that the CRISPR–Cas system could be used for human gene therapy”

Together, these studies suggest that the CRISPR–Cas system could be used for human gene therapy to correct genetic defects not only in affected patients but also in their germline, which ensures that their progeny will also lack the disease.