The recent development of custom designed nucleases, such as Zinc-Finger Nucleases (ZFN), Transcription Activator-Like Effector Nucleases (TALENs) and the Clustered Regularly Interspaced Short Palindromic Repeat Associated system (CRISPR/Cas9) has opened up exciting opportunities to edit genomes in a wide range of organisms (Joung and Sander, 2013 for review). Knocking out protein-coding genes can be easily achieved by using just one pair of such dimeric nucleases, to target the first coding exon, thereby introducing short indels that result in a translational frameshift. Several reports have also demonstrated the possibility to target larger genomic rearrangements by using two pairs of nucleases (Carlson et al., 2012; Gupta et al., 2013; Lee et al., 2011). Although homologous recombination mediated genetic engineering is feasible in some systems, such as mouse embryonic stem cells, this approach requires multiple steps, including the selection of drug-resistant clones, and can be laborious depending on the target and nature of the targeting. This approach is being rapidly superceded by the advent of custom ZFN, TALEN and CRISPR/Cas9 technologies, which enable the extremely rapid and efficient disruption of not only coding, but also non-coding elements, by creating deletions, or by changing local genomic organization by creating inversions.
Institut Curie, 26 rue d'Ulm 75005 Paris France
Corresponding author: Elphège P Nora, Edith Heard
Comments from Claire Rougeulle
We are currently using these protocols for targeted deletion in human ES cells but we are still in the middle of the process so we cannot comment on the differences between the two cell systems.
There are however few general questions and some minor comments.
- what is the smallest deletion that can be engineered using these systems (TALENs or CRISPR/Cas9) (related to note A, Page 3).
- It could be interesting to discuss off target effects, especially for CRISPR/Cas9: how to control for this, how many pairs of endonuclease should be used to eliminate non-specific effects.
- Page 4, last paragraph: typographic error: through
- Page 5, point 8: likely 1/10 000, and not 1/1000 as in 7)
- Page 7, note A: using elongation time allowing for amplifying the undeleted form in the case of small deletion would be useful as internal positive control (this fits with note A page 10)
- Page 7, note E: would it be possible to elaborate on the use of the T7 endonuclease I assay. In our hands, the results were not very informative: the cleavage product was barely visible on gel whereas the deletion, tested by PCR, worked very efficiently
- Page 8: we usually split the 96 well plates in three: one will be used for genotyping, two for maintaining cells in culture (and freezing is necessary). If cells are feeder dependent, we plate a lower dilution (1/10 instead of 8/10) of cells in the genotyping plate to avoid contamination from feeders coming from the original plate
- Page 10 note C: PCR positive controls should be included in the initial PCR analysis. In the case of a small deletion, using an appropriate elongation time allow for amplifying the undeleted allele. In the case of a long deletion, a previous sample in which the deletion worked can be used as a positive control is such a sample is available. Otherwise, we recommend to artificially design a construct containing template DNA mimicking the deletion product