Marvin Yingbin Ouyang discusses in vivo gene editing via the power of TALEN and CRISPR/Cas9.
The gold standard for genetically engineering mouse models is ES-cell based homologous recombination. However, this approach is time-consuming and costly. A novel mouse model can take up to two years and cost >US$50,000. Recently, TALEN and CRISPR/Cas9 systems have been harnessed to edit genomes of cultured cells, mice and rats(1),(2). Both TALEN and CRISPR/Cas9 enable genome editing, but have different advantages (Fig. 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. Both systems create double strand breaks at target sites, which are repaired by non-homologous end joining (to create knockouts), or homologous recombination in the presence of homologous repair template (to introduce point mutations or insertions). The following should be considered when selecting an approach.
Off-target effects
A disadvantage of nuclease-based genome editing is potential cleavage at undesired locations. Although off-target effects have been reported using CRISPR/Cas9 in human cell lines(3), analyses of CRISPR/Cas9 knockout mice suggest lower off-target frequency in vivo4. It is reported that using paired nickase or truncated sgRNAs (17 nucleotides instead of 20) improves specificity(5), (6). Studies suggest these strategies reduce genome editing efficiency, and it is unclear whether in vivo off-target effects are reduced.
Instead of the single gRNA guiding Cas9, TALEN pairs bind opposite sides of the target site, separated by a 14-20 nucleotide spacer. Since FokI functions as a homodimer, TALEN off-target frequency may be lower than CRISPR/Cas9(7).
Efficiency
Both systems enable efficient genome editing in vitro and in vivo. Modifications can
be introduced by directly injecting RNAs encoding Cas9 protein and gRNA, or TALENs, into one-cell stage fertilised eggs(4),(8). This eliminates laborious targeting vector construction and ES cell gene targeting required to generate knockout mice using traditional techniques. High CRISPR/Cas9 efficiency means mutations can be introduced at multiple loci by injecting multiple gRNAs with Cas9(4).
Target sites
The requirement for a PAM (NGG) sequence immediately preceding the target sequence limits site selection for CRISPR/Cas9(9). Since either DNA strand can be targeted, this is no barrier for gene knockout, but may present difficulties in site-specific mutations or knockins. In contrast, TALENs can be generated to specifically target nearly any sequence in the genome.
Target design and construction
Because target specificity for CRISPR/Cas9 relies on RNA/DNA hybridisation rather than protein/DNA interaction, gRNAs are simpler to construct than TALENs, taking only one to three days to construct a gRNA vector. However, currently available TALEN recognition modules have greatly reduced work required to clone TALEN vectors.
As both TALEN and CRISPR/Cas9 systems show great promise in vitro and in vivo – 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. ES cell gene targeting remains important for the generation of conditional and inducible alleles.
Cyagen Biosciences is a contract research organisation that generates custom, genetically modified mouse and rat models by ES cell homologous recombination, TALEN, and CRISPR/Cas9 methods. The organisation serves pharmaceutical companies and academic bioscience research labs worldwide. Its range of services include mouse and rat transgenic, knockout, knockin, humanisation, and nuclease-mediated genome editing.
References:
1 Gaj T, Gersbach CA, Barbas CF 3rd (2013) ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol. 31: 397-405
2 Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelsen TS, Heckl D, Ebert BL, Root DE, Doench JG, Zhang F (2014) Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343: 84-87
3 Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK, Sander JD (2013) High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol. 31: 822-826.
4 Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153:910-918
5 Cho SW, Kim S, Kim Y, Kweon J, Kim HS, Bae S, Kim JS (2014) Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res. 24: 132-141
6 Fu Y, Sander JD, Reyon D, Cascio VM, Joung JK (2014) Improving CRISPR-Cas nuclease specificity using truncated guide RNA. Nat Biotechnol. 32: 279-284
7 Park CY, Kim J, Kweon J, Son JS, Lee JS, Yoo JE, Cho SR, Kim JH, Kim JS, Kim DW (2014) Targeted inversion and reversion of the blood coagulation factor 8 gene in human iPS cells using TALENs. Proc Natl Acad Sci USA 111: 9253-9258
8 Tesson L, Usal C, Ménoret S, Leung E, Niles BJ, Remy S, Santiago Y, Vincent AI, Meng X, Zhang L, Gregory PD, Anegon I, Cost GJ (2011) Knockout rats generated by embryo microinjection of TALENs. Nat Biotechnol. 29: 695-696
9 Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, Peterson RT, Yeh JR, Joung JK (2013) Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol. 31: 227-229
For more information visit www.scientistlive.com/eurolab
Marvin Yingbin Ouyang is with Cyagen Biosciences in the USA. www.cyagen.com
