Genetic Engineering in the Press by GEG

Principal editing (PE) is thought to be a safer and more precise alternative to CRISPR-Cas9. Using a Cas9 nickase and reverse transcriptase, master editors can create small insertions and deletions without creating double-strand breaks in the DNA and without requiring a donor template for repair. There are four different types of master editing systems. PE1 and PE2 use a Cas9 nickase fused to a reverse transcriptase and a master editing guide RNA (pegRNA). PE3 and PE3b are derivatives that use the PE2 system in conjunction with an additional single guide RNA (sgRNA). Master editing was tested in hereditary tyrosinemia, an autosomal recessive disease caused by a loss-of-function mutation in the fumarylacetoacetate hydrolase (Fah) gene. The pathogenic mutation prevents patients from producing the enzyme fumarylacetoacetate hydrolase, which contributes to the normal breakdown of the amino acid tyrosine. A research team delivered PE3 components by hydrodynic injection or PE2 using adenovirus in a mut-mut FAH mouse model. Compared to the original Cas9 which induces double-strand breaks, main editing, in particular PE2, was shown to be a safer strategy in terms of off-target effects. Extensive analyses showed that the off-target effects of master editing were less than 0.1%.

Despite spooking investors, new insights into DNA repair and the CRISPR gene-editing system are part and parcel of its progress from research tool to human therapy.

A team of Chinese scientists has conducted the first full assessment of the effects of CRISPR on non-human primates. The study set out to study whether the CRISPR-Cas9 genome editing technique results in off-target mutations in rhesus macaque monkeys.

The study has not been officially published or peer-reviewed at this stage so it shouldn't be declared as entirely reliable, but it does offer promise that the CRISPR-Cas9 process is relatively accurate.

Scientists have discovered a virus-made protein that can block the powerful gene-editing tool CRISPR-Cas9 from cutting DNA. The protein allows researchers to better control CRISPR so that it doesn’t snip unintended pieces of genetic code. In the future, the technique could be used to make gene editing more precise — and safe.

As CRISPR-Cas9 starts to move into clinical trials, a new study published in Nature Methods has found that the gene-editing technology can introduce hundreds of unintended mutations into the genome.

Quantifying the outcome of CRISPR editing experiments to assess unanticipated events, especially for clinical use of the technology is essential but existing tools are not sensitive enough. These tools still cannot easily detect translocations without performing cumbersome experiments. However, for the first time, unwanted translocation events can be detected in multiplex PCR experiments after CRISPR editing thanks to a new software tool, CRISPECTOR that effectively separates signal from noise and can detect, statistically evaluate and quantify many types of off-target genome editing activities. Thus, this software learns to recognize specific patterns of background noise from next-generation sequencing read errors and incorrect nucleotide insertion during multiplex PCR reactions. Moreover, one experiment showed that a high rate of 1-bp deletions at the expected cut site masks the actual editing signal in tools using subtraction only. In this case, CRISPECTOR overcame the high background and estimated an editing activity of 0.113% while other software such as CRISPResso2 and ampliCanb estimated much lower signals at 0.068% and 0.07%, respectively.

Among the most significant scientific advances in recent years are the discovery and development of new ways to genetically modify living things using a fast and affordable technology called CRISPR. Now scientists at The University of Texas at Austin say they’ve identified an easy upgrade for the technology that would lead to more accurate gene editing with increased safety that could open the door for gene editing safe enough for use in humans.

Scientists at the University of Alberta in Canada have developed a technology that can dramatically improve the specificity of CRISPR-Cas9 gene editing. The approach uses synthetic guide molecules known as bridged nucleic acids (BNAs) in place of the system’s native guide RNAs (gRNAs) to direct the Cas9 enzyme to its target DNA sequence, and so reduce off-target DNA cleavage.

Scientists at the University of California, Berkeley and Massachusetts General Hospital have identified a key region within the Cas9 protein that governs how accurately CRISPR-Cas9 homes in on a target DNA sequence, and have tweaked it to produce a hyper-accurate gene editor with the lowest level of off-target cutting to date.

Discarded next-gen sequencing chips provide a platform for analyzing protein-DNA interactions
that reveals a novel proofreading mechanism used by the Cascade/Cas3 complex.

Researchers say they discovered hundreds of mutations during a gene editing experiment, casting doubt on CRISPR's safety and precision.