The gold standard for genetically engineering mouse models is ES-cell based homologous recombination. However, this approach is very time-consuming and costly. Recently, TALEN and CRISPR/Cas9 systems have been harnessed to edit genomes of cultured cells, mice and rats1,2. Both systems can be used to create knockouts, and to introduce point mutations or small insertions, but each has distinct advantages (see Table 1). TALENs are chimeric proteins composed of site-specific DNA-binding domains fused to the non-specific endonuclease FokI. CRISPR/Cas9 uses a site-specific single guide RNA (sgRNA) to direct the Cas9 nuclease to its target locus.
|Origin||Plant pathogenic bacteria (Xanthomonas)||Diverse bacteria|
|Components||Pairs of TALE-FokI fusion proteins||Guide RNA and Cas9|
|Efficiency||High||High but variable|
|Off-target effects||Minor||Moderate to high|
|Target site availability||No restriction||Requires PAM (NGG) motif|
|Time required for vector engineering||One week||1-3 days|
Because TALEN pairs bind opposite sides of the target site, TALEN-mediated cleavage at other sites in the genome is unlikely4. In contrast, off-target effects have been reported using CRISPR/Cas9 in cell lines3, but analyses of CRISPR/Cas9 knockout mice suggest lower off-target frequency in vivo5.
Genome modifications can be introduced by directly injecting RNAs encoding TALENs or Cas9 protein and gRNA, into one-cell stage fertilized eggs5,6. Mutations can also be introduced at multiple loci by coinjecting multiple gRNAs with Cas94.
TALENs can be generated to specifically target nearly any sequence in the genome. In contrast, target site selection for CRISPR/Cas9 is limited by the requirement for a PAM (NGG) sequence7. Since either DNA strand can be targeted, this is no barrier for gene knockout, but may present difficulties in site-specific mutations or insertions.
Because targeting of CRISPR/Cas9 relies on simple RNA/DNA hybridization, gRNAs are easier to design and construct than TALENs, taking only 1-3 days. However, currently available TALEN recognition modules have greatly reduced work required to clone TALEN vectors.
Both TALEN and CRISPR/Cas9 systems show great promise- Which approach should you choose? To generate a single- or double-knockout quickly, try CRISPR/Cas9. Otherwise, TALEN offers fewer off-target effects and target sequence requirements. Unfortunately, for the generation of conditional and inducible alleles, ES cell-based gene targeting remains the only option.
Cyagen offers a complete line of genome editing services using TALENs and CRISPR/Cas9, in both mice and rats. Visit our site to learn more about our rapid and cost-effective genome editing options.
If you are want to use nuclease-mediated genome editing in your own experiments, but aren't interested in animal models, custom TALEN vectors are also available from VectorBuilder.com.
Speak to our specialists:
Tel: (800) 921-8930
TurboKnockout® Gene Targeting - ES-based knockout mice, 100% guaranteed germline transmission, as fast as 6 months
CRISPR Knockout - Guaranteed germline transmitted F1 animals, as fast as 3 months
Transgenic Mice - More consistent expression, defined region of integration, founders as fast as 3 months