Choosing a system for in vivo genome editing: Is TALEN or 타겟 유전자 편집 best for your project?
유전자 편집 기술 개요
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 타겟 유전자 편집 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. 타겟 유전자 편집 uses a site-specific single guide RNA (sgRNA) to direct the 타겟 유전자 편집 nuclease to its target locus.
TALEN과 타겟 유전자 편집 비교
| TALEN | 타겟 유전자 편집 | |
|---|---|---|
| Origin | Plant pathogenic bacteria (Xanthomonas) | Diverse bacteria |
| Components | Pairs of TALE-FokI fusion proteins | Guide RNA and 타겟 유전자 편집 |
| 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 |
| Multiplexing | Moderate | Efficient |
Off-target effects
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 타겟 유전자 편집 in cell lines3, but analyses of 타겟 유전자 편집 knockout mice suggest lower off-target frequency in vivo5.
Efficiency and multiplexing
Genome modifications can be introduced by directly injecting RNAs encoding TALENs or 타겟 유전자 편집 protein and gRNA, into one-cell stage fertilized eggs5,6. Mutations can also be introduced at multiple loci by coinjecting multiple gRNAs with 타겟 유전자 편집4.
Target sites
TALENs can be generated to specifically target nearly any sequence in the genome. In contrast, target site selection for 타겟 유전자 편집 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.
Target design and construction
Because targeting of 타겟 유전자 편집 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 타겟 유전자 편집 systems show great promise- Which approach should you choose? To generate a single- or double-knockout quickly, try 타겟 유전자 편집. 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.
서비스 및 문의
Cyagen offers a complete line of genome editing services using TALENs and 타겟 유전자 편집, 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
[email protected]
원스톱 마우스 모델 검색 플랫폼: MouseAtlas
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추가 정보 및 참고 문헌
- Gaj T, Gersbach CA, Barbas CF 3rd (2013) ZFN, TALEN, and 타겟 유전자 편집/Cas-based methods for genome engineering. Trends Biotechnol. 31: 397-405
- 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 타겟 유전자 편집 knockout screening in human cells. Science 343: 84-87
- Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK, Sander JD (2013) High-frequency off-target mutagenesis induced by 타겟 유전자 편집-Cas nucleases in human cells. Nat Biotechnol. 31: 822-826.
- 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
- 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 타겟 유전자 편집/Cas-mediated genome engineering. Cell 153:910-918
- Tesson L, Usal C, M\A8\A6noret 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
- 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 타겟 유전자 편집-Cas system. Nat Biotechnol. 31: 227-229
