A California-based 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.
Integrated DNA Technologies (IDT) said the Alt-R S.p. HiFi Cas9 Nuclease 3NLS enzyme 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 off-target effects without significant loss of on-target activity, according to IDT.
The 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.
For example, genome editing could inadvertently disable a tumour-suppressor gene or activate a cancer-causing gene. Or, consider the possibility of an off-target effect where two different chromosomes are joined in a phenomenon called translocation. 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 – with results that 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 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 on-target 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.
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The largely preferred method of delivering genome editing reagents as RNP complexes reduce, but does not eliminate, the risk of off-target editing.
“Previous attempts to make improved specificity Cas9 mutants focused on plasmid-based methods, which greatly overexpress the Cas9 protein and maximize unwanted side effects,” said Mark Behlke, chief scientific officer at IDT. “To achieve reduced off-targeting in the face of sustained overexpression, this first generation of Cas9 variants relied on mutations that compromised activity, which in turn led to poor function when used in RNP format.”
“IDT specifically developed a mutant that performs well when used with the lower levels of protein employed with RNP delivery, maximizing safety and further reducing unwanted side effects,” said Behlke.