Improved CRISPR

The use of CRISPR techniques to cleave and cut/add DNA sequences is a two step process. First the use of a CRISPR or equivalent to target the sequence and then the CAS9 to cut it.  Target and cut. However there are problems. The target may appear at multiple locations and the DSB, double stranded break cut, is often subject to faulty repairs. Thus, although CRISPER can be an elegant approach, its errors can have massive defects.

Doudna and her team have announced some improvement in Nature. They state:

The RNA-guided CRISPR–Cas9 ... has been widely repurposed for genome editing. High-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9...) variants exhibit substantially reduced off-target cleavage in human cells, but the mechanism of target discrimination and the potential to further improve fidelity are unknown. Using single-molecule Förster resonance energy transfer (smFRET) experiments, we show that both SpCas9-HF1 and eSpCas9... are trapped in an inactive state when bound to mismatched targets. We find that a non-catalytic domain within Cas9, REC3, recognizes target complementarity and governs the HNH nuclease to regulate overall catalytic competence. Exploiting this observation, we designed a new hyper-accurate Cas9 variant (HypaCas9) that demonstrates high genome-wide specificity without compromising on-target activity in human cells. These results offer a more comprehensive model to rationalize and modify the balance between target recognition and nuclease activation for precision genome editing.

As  The Scientist notes:

To minimize the off-target effects of CRISPR-based genome editing, researchers have designed a version of Cas9, the enzyme that cuts DNA, that avoids mistakes with unprecedented precision. Mutating one piece of the nuclease, called REC3, reduced off-target effects to below that of other high-fidelity Cas9 enzymes....namely, eSpCas9(1.1) and SpCas9-HF1—are more specific to their target sequences and make fewer off-target cuts than the natural version. That’s when they identified REC3’s role in sensing the accuracy with which the enzyme binds to an intended stretch of DNA: in the event of a mismatch, the team found, REC3 would keep Cas9 inactive. ...When they tested this new HypaCas9 on six target sites, the researchers found that at two sites the nuclease made no mistakes, at three sites it made one error, and for one site it hit 18 unintended loci. In comparison, wild-type Cas9 only had a perfect score once, and otherwise tallied between eight and 134 off-target effects, depending on the intended site. The two more-specific Cas9 variants performed just as well as HypaCas9 for five sites, but had a few additional errors at the sixth.

 

 This may be a significant step forward in improving CRISPR usage and safety.