genome
| By Nestor Arellano
Cas9 enzyme variant cuts down CRISPR off-targets On-target editing was just as good as wild-type Cas9 and off-target events were greatly reduced
T
he ability to modify the gene of an organism through CRISPR genome editing holds the promise of curing diseases such as cancer and leukemia. However, there are also growing concerns that the innovative gene editing technology could alter regions of the genome which researchers are not targeting. A custom manufacturer of DNA and RNA oligonucleotides has launched a Cas9 enzyme variant which it said can drastically reduce off-target effects in CRISPR genome editing. The Alt-R S.p. HiFi Cas9 Nuclease 3NLS enzyme developed by Integrated DNA Technologies (IDT) is a recombinant S. pyogenes Cas9 mutant that improves specificity while maintaining a high editing efficiency similar to wild-type Cas9. The Cas9 enzyme variant is able to cut down on off-target effects without significant loss of on-target activity, according to IDT. Off-target gene editing could have serious consequences. For example, genome editing could inadvertently disable a tumour-suppressor gene or activate a cancer-causing gene. There is also the possibility of an off-target effect where two different chromosomes are joined in a phenomenon called transloca-
tion. Translocation is the cause of chronic myeloid leukemia and other conditions. The new enzyme has been tested at a number of prominent laboratories conducting translational research into various diseases.Results have exceeded all expectations, according to a press release from IDT. “We performed an unbiased evaluation of several versions of high-fidelity Cas9 enzyme in primary human stem cells,” said Dr. Matt Porteus from the Stanford University’s
TAble 1 IDT Cas9 enzyme
24 BIOTECHNOLOGY FOCUS August/September 2017
Division of Stem Cell Transplantation and Regenerative Medicine. “We have been very impressed with the characteristics of this new IDT enzyme.” He said that unlike other versions, Alt-R S.p. HiFi Cas9 Nuclease 3NLS consistently achieved high on-target editing activity while having low off-target activity. “Because of the retained excellent ontarget activity and improved specificity profile, we are excited to use this version in our future experiments focused on developing novel genome editing based therapies for severe diseases with unmet medical needs,” said Porteus. In order to successfully provide a Cas9 mutant with radically reduced off-target effects while maintaining high on-target activity, IDT screened more than 250,000 mutants in two rounds of selection. The resulting rigorously tested enzyme, Alt-R S.p. HiFi Cas9 Nuclease 3NLS, is further enhanced with three nuclear localization signals (NLS) for optimal migration to the target DNA. A few months ago, Dr. J. Keith Joung of Massachusetts General Hospital, showed some 150 experts from biotech industry and academia an example where CRISPR is supposed to edit the VEGFA gene on chromosome 6. VEGFA stimulates the production
