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CAS9 ENzYME VARIANT CUTS DowN CRISPR oFF-TARGETS
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.
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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 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.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 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
table 1 idt CaS9 enzyme
CaS9 gene editing
of blood vessels, including those used by cancerous tumors.
“Although each CRISPR has zero to a dozen or more ‘known’ off-target sites (where known means predicted by those web-based algorithms),” Joung said, “there can be as many as 150 ‘novel’ off-target sites, meaning scientists had no idea those errors were possible.”
Off-target effects occur because CRISPR has two parts. The RNA part targets the site in the genome specified by the RNA’s string of nucleotides. The enzyme cuts the genome at this site.
However, a genome can have more than one site where the same string of nucleotides appears.
The cutting enzyme of CRISPR does not stop at one cut. The enzyme “still has the energy to bind with an off-target site, so it can still cleave those sites,” said Joung.
alternative to CriSpr genome editing
IDT is not the only company interested in developing a solution to CRISPR Cas9 offtarget effects.
Before IDT’s release of its Cas9 enzyme variant, chemical and life sciences firm MilliporeSigma reported that it developed an alternative to CRISPR genome editing.
The new technique, called proxy-CRISPR, provides researchers with more experimental options and rapid results that can help speed up drug development, according to Udit Batra, CEO of the company.
“With more flexible and easy-to-use genome editing technologies, there is greater potential in research, bioprocessing, and novel treatment modalities,” he said. “… MilliporeSigma’s new technology is just one example of our commitment to solving challenges in the genome editing field, and we will continue to make CRISPR research a priority.”
Most natural CRISPR systems, found in bacteria, cannot work in human cells without significant re-engineering.
However, proxy-CRISPR provides a rapid and simple method to increase their usability without the laborious need to re-engineer native CRISPR proteins, according to Batra.
MilliporeSigma has filed several patent applications on the proxy-CRISPR technology.
Other scientists have expressed confidence in IDT’s Cas9 variant enzyme.
“The IDT high fidelity Cas9 performed admirably in primary human hematopoietic stem cells. On-target editing was just as good as wild-type Cas9 and off-target events were greatly reduced,” said Dr. Jacob Corn, director of the Innovative Genomics Initiative at the University of California Berkeley.
The typically preferred method of delivering genome editing reagents as RNP complexes do not eliminate the risk of off-target editing.
“Previous attempts to make improved specificity Cas9 mutants focused on plasmidbased 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.”
To see this story online visit https://biotechnologyfocus.ca/idt-launchescas9-enzyme-which-cuts-down-crispr-offtargets/
