Zinc-finger nucleases (ZFNs) are engineered proteins which combine the highly sequence-specific DNA binding ability of multimeric zinc-finger protein domain, where individual zinc-finger motifs capable of binding triplets of DNA sequence are linked together, with the nuclease activity of the restriction endonuclease Fok
. The plasticity of the zinc-finger domain allows for the design of ZFNs which can bind specifically to a broad range of sequences 19, 20
. Libraries of zinc-finger motifs have been engineered with sufficient complexity that ZFN reagents can be designed to target most user-defined sequences. The nuclease domain functions to cleave the target sequence. Because the nuclease domain must form a dimer to cleave double stranded DNA, two ZFNs are needed to target a specified sequence. When introduced into a cell, the two ZFNs bind to their respective binding sites, positioning their nuclease domains to interact and introduce a site-specific double-strand break (DSB) in the chromosome. This break is subsequently repaired by the cell using either the highly-conserved homology-dependent repair (HDR), or non-homologous end joining (NHEJ) DNA repair pathways 18
. In the case of HDR, the sequence is repaired precisely and the ZFN heterodimer can reform on the repaired target sequence and re-cut. Since NHEJ-mediated repair is less accurate than HDR, it occasionally results in the loss or addition of nucleotides, resulting in a mutation 21
. This mutation, in turn, often results in a frameshift in the gene coding sequence, leading to a truncated and/or nonsense peptide. Beginning with pioneering work in the fruit fly 22
, engineered ZFNs have been used to generate site-specific mutations in a variety of cells and embryos from several species 23, 24
Initial experiments applying ZFNs to the rat produced site-specific mutations in genes with a surprisingly high efficiency 25, 26
. ZFNs can be used to produce heritable, site-specific targeted mutations in the rat by combining in vitro
transcribed ZFN-encoding nucleic acids to the one-cell embryo via standard transgenic microinjection techniques (Box 2
). Action of the ZFNs during the earliest cell divisions leads to a high percentage of modified chromosomes in the resulting offspring. Modified alleles are transmitted through the germ-line and can be backcrossed to establish multiple strains with unique mutant alleles.
Box 2. Gene knockout via ZFNs
Zinc-finger nucleases are engineered proteins which are designed to target and disrupt genes when introduced into a cell by inducing a double strand break in the chromosome. Innate cellular DNA repair mechanisms repair the break, although frequently deleting small bits of sequence. When the ZFNs induce a break in the coding sequence of a gene, the deletion created during repair disrupts the function of that gene. ZFNs can be injected into the rat embryo in either plasmid DNA or mRNA form to disrupt a target gene. Thus far, commercially developed reagents have been successful in knocking out genes in the rat 25, 26
, although academic sources of reagents are demonstrating improved utility 57, 58
and may be equally applicable to the rat. ZFN mRNA is injected into the rat embryos derived from superovulated females using standard pronuclear injection techniques, although cytoplasmic injection can also work 25, 27
(). Injected embryos are transferred to pseudopregnant females and the resulting offspring are screened for ZFN activity using a CEL I nuclease assay, which is based on the affinity of this enzyme for heteroduplex (mismatched) DNA 27
. ZFN-modified founder animals are bred to assay for inheritance of the germline modification and to create uniformly modified F1 rats. About one-third of the mutations will result in an in-frame deletion or insertion that may or may not disable the gene. For this reason, characterization of ZFN-induced alleles by sequencing to identify desirable alleles is essential prior to attempting germline transmission.
Figure I ZFNs can be applied to the rat embryo to knock out genes. The engineered reagents are designed, assembled and tested in vitro, either commercially or by the user. Active ZFN pairs are introduced by pronuclear mciroinjection and transferred to pseudopregnant (more ...)
There are three major advantages of this strategy. First, it is very rapid, in the order of months. Second, it has worked in all strains tested to date 26–28
. Third, it does not result in the incorporation of exogenous DNA. One current limitation of ZFNs relates to design for small genes or closely related gene families due to the requirement of unique sequences for which ZFNs can be assembled from the existing libraries, both of which could hamper selective design. A second limitation is a potential, albeit rare, off-target effect where ZFNs cause DSBs and mutations at undesired loci. Such effects were noted in the application of ZFNs to the zebrafish 29, 30
, but were not found to be prevalent in either rat study 25, 26
. Nevertheless, the potential effects of rare off-target events would be easily mitigated by backcrossing, resulting in the loss of any potential undesired mutations.
Since it was first reported, two other groups have reported rat gene knockouts using this approach (26
) and we have knocked out 54 genes ourselves in a span of 10 months. The protocol has been highly efficient, requiring injection of an average of 297 embryos per target gene (range 71–1283), screening an average of 29 live born pups (range 3–208) and has resulted in an average of 3.5 knockout founders per gene (range 1–14) (PhysGen Knockout Team, unpublished). The knockout of this many target genes demonstrates the reproducibility of the approach and also that the current commercially owned libraries of ZFN reagents are sufficient to target and knockout most, if not all genes in the rat genome. The approach is also very rapid, as demonstrated by the publication of an X-linked severe combined immunodeficiency (X-SCID) rat model by knocking out the interleukin 2 receptor gamma with very high efficiency 26
. From target site selection to published homozygous knockout phenotype, this study took approximately 12 months (personal communication). This speed and ease is unprecedented and opens up entirely new research opportunities for investigators interested in using rats. The Sigma Advanced Genetic Engineering (SAGE) Lab also recently applied the same approach to mice 31
. Other potential hurdles to ZFN engineering in the rat include the cost ($25,000 USD) and associated licensing agreement ($7,500 USD) associated with the commercial ZFNs used in the rat studies. We have recently discussed these issues and alternative ZFN sources in another recent review 23
, but add here that engineering a knockout mouse can cost as much as $100,000 USD and take a whole year to generate a single animal 32
. Thus, considering the speed and reproducibility, the ZFN method is a cost effective way to generate novel knockout rat models.
Finally, as HDR is a cellular repair mechanism for a DSB which is active in most cells, it is possible to provide a template in the form of an exogenous DNA fragment so that the HDR mechanism results in precise incorporation, or ’knockin’ of the template. Several groups have now used ZFNs to facilitate targeted integration of new sequences into genomes by stimulating the HDR mechanism. To date, ZFNs have been used for knockin of a few base pairs up to 9-kilobase expression constructs into Drosophila
embryos and cultured human cells 22, 33–39
and, recently, ZFNs were used to target expression cassettes into human embryonic- and induced pluripotent stem cells 40, 41
. It will be very interesting to see if similar approaches can be routinely applied to knock new sequences into the rat embryo genome by microinjection. Gene knockin in the rat embryo would potentially allow for many exciting new approaches including the generation of conditional gene alleles to allow for temporal and/or spatial ablation of gene function in different cells and tissues as has been done routinely in the mouse 42