Cas9 has already been shown to be able to knock out up to kilobase-size fragments of DNA, making it a big hitter in the world of genome editing. But one of the biggest enemies for researchers using such tools is the diploid karyotype, which impairs the study of the effects of a mutation or of a gene deletion due to the compensatory role of its additional copy.
To overcome this opponent, a team led by Dr Tilmann Bürckstümmer generated a haploid human cell line, using the hard-hitting power of Cas9, and in the process pushed the boundaries of CRISPR/Cas9 knockout ability.
- The researchers started with the HAP1 fibroblast-like cell line, which had been previously established from the reprogramming of the nearly haploid chronic leukemia derived cell line KBM-7.
- They designed Cas9 guiding RNA targeting each one of the insertion sites of Chromosome 15 into Chromosome 19, the only disomic portion of HAP1’s genome, which contains over 300 genes spread within 30 million base pairs.
- This resulted in successful simultaneous deletion of the massive fragment and the creation of the haploid cell lines ‘eHAP’ (short for ‘engineered haploid’).
- The Austrian team achieved an efficiency of approximately 1%; to put this in perspective, a similar challenge with TALEN had an efficiency of approximately 0.5%.
This stunning feat shows that Cas9 has abilities beyond simple gene knockouts, and could be used in the deletion of large regulatory regions such as enhancers and promoters.
Furthermore, if this did not score high enough on the board, the newly established cell line was shown to maintain the haploid state in culture and DNA sequencing revealed no off-target effects of Cas9 and only some mutations, attributed to genetic drift. Lastly, RNA sequencing revealed no differences between the parental cell line and eHAP, apart from the ones arising from the deleted region.
This study generates a useful tool to study the effects of single gene deletions and could represent, as the authors conclude, the starting point to define a minimal-essential genome.
For the full details of this exciting match check out the full paper at Genome Research, November 2014.