112 GENOME EDITING


In vivo genome editing T


Marvin Yingbin Ouyang discusses in vivo gene editing via the power of TALEN and CRISPR/Cas9.


he 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 rats1, 2


. Both TALEN and


Fig. 1. Comparison of TALEN and CRISPR/Cas9 technologies.


CRISPR/Cas9 enable genome editing, but have different advantages (Fig. 1).


TALEN Origin


Components Efficiency Off-target effect Target site availability


Time required for vector engineering Multiplexing


Plant pathogenic bacteria (Xanthomonas)


TALE-Fokl fusion protein High Minor No restriction


One week Moderate


Fig. 2. Cyagen Biosciences AAALAC- accredited animal facility provides a specific pathogen-free environment for custom animal model generation.


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). Te following should be considered when selecting an approach.


Off-target effects A disadvantage of nuclease-based genome editing is potential


CRISPR/Cas9 Diverse bacteria


Guide RNA and Cas9 High but variable Moderate to high Requires PAM (NGG) motif


1-3 days Efficient


cleavage at undesired locations. Although off-target effects have been reported using CRISPR/ Cas9 in human cell lines3


,


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 specificity5, 6


frequency may be lower than CRISPR/Cas97


. 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 eggs4, 8


.


Tis 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 Cas94


.


Target sites Te requirement for a PAM (NGG) sequence immediately preceding the target sequence limits site selection for CRISPR/Cas99


. 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.


. 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


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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


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