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MilliporeSigma's New CRISPR System Moves Chromatin Out of Way for More Efficient Editing

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NEW YORK – Even as some biotechnology companies are inching closer to developing CRISPR-Cas-based gene editing therapeutics for a variety of human diseases, researchers are still finding ways to make the technology safer and more efficient.

While some have chosen to go the route of optimizing the nucleases themselves — whether by developing proteins from scratch or by engineering orthologs of existing proteins that recognize larger regions of the genome, for example — Merck KGaA life science business MilliporeSigma has opted for a slightly different strategy. The company has developed a technology called CRISPR-chrom, which fuses chromatin-modulating peptides to CRISPR proteins. Essentially, this complex moves the chromatin out of the way, allowing the CRISPR protein to reach its target more efficiently.

MilliporeSigma was awarded a patent for CRISPR-chrom earlier this month, its second in the CRISPR space in the US. The company said it is the only firm in the US with intellectual property in the CRISPR chromatin field. Its first CRISPR patent in the US was awarded in February 2019 for a similar technology called proxy-CRISPR, which was published in a Nature Communications study in June 2017.

In that study, the researchers had sought to address the problem of CRISPR proteins being blocked from reaching human cells in order to edit them. They began by looking at the widely adopted type II-A Streptococcus pyogenes Cas9 (SpCas9) in order to determine whether there was a way to eliminate its off-target effects and possibly create a strategy for selectively editing identical genomic sites in different genes within the same genome. In conducting their research, they found that human chromatin proteins were blocking CRISPR biomolecules from reaching human DNA.

In exploring other CRISPR-Cas systems that could work more precisely than SpCas9, the team identified the type II-B FnCas9 from the Francisella novicida bacterium, noting that it possessed a novel enzymatic property that cleaved target DNA in a staggered pattern, exhibiting a higher intrinsic specificity than SpCas9. So, they developed proxy-CRISPR, which restored FnCas9 nuclease activity in a target-specific manner and was applicable to a variety of CRISPR-Cas systems.

Importantly, proxy-CRISPR enhanced the usability of native CRISPR proteins without the need to re-engineer them. The researchers also hypothesized that the method could be used to reduce the off-target effects of genome editing.

While the company is pleased with proxy-CRISPR's effectiveness, "we envisioned an alternative approach to moving chromatin, which we call CRISPR-chrom," Angela Myers, MilliporeSigma's head of genome editing and novel modalities, told GenomeWeb in an email interview. "Chromatin is one of the last molecular barriers that CRISPR proteins have to navigate prior to accessing the genome for editing."

The company first described CRISPR-chrom in a February 2019 study in The CRISPR Journal. The researchers showed that modifying SpCas9 by fusing it with chromatin-modulating peptides (CMPs) derived from high mobility group proteins HMGN1 and HMGB1, histone H1, and chromatin remodeling complexes, improved its activity by up to several fold, particularly on refractory target sites. They also showed that the CMP fusion strategy was effective in improving the activities of smaller Cas9 nucleases from Streptococcus pasteurianus and Campylobacter jejuni, as well as four newly characterized Cas9 orthologs from Bacillus smithii, Lactobacillus rhamnosus, Mycoplasma canis, and Parasutterella excrementihominis.

In fact, they noted, it was their work with developing proxy-CRISPR that led to their development of CRISPR-chrom. "We previously found that the inactivity of certain Type II-B and Type II-C Cas9s on certain targets could be reversed by binding of a catalytically inactive SpCas9 to proximal locations of the target site, a scheme that we referred to as proxy-CRISPR," the authors wrote. "This finding led us to reason that fusion modification of Cas9 protein with peptides known to interact with chromatin naturally might also enhance Cas9 activity in the context of chromatin."

Indeed, the researchers found that among the peptides that possess native DNA binding activities, the human HMGB1 box A, the human histone H1 central globular domain, and the DNA binding domains of yeast chromatin remodeling complexes ISWI and CHD1 increased SpCas9 activity by 1.7- to 2.5-fold, compared to unmodified SpCas9. They then tried to further augment the fusion effect by modifying SpCas9 on both the amino and carboxyl termini with HMGN1, HMGB1 box A, histone H1 central globular domain, and ISWI and CHD1 DNA binding domains in 16 combinations, and found that the three top-ranking double-fusion nuclease combinations increased the activity by 2.5- to 3.4-fold compared to the unmodified SpCas9.

According to Myers, "the products and services we are developing from CRISPR-chrom will involve Cas9-CMP fusion proteins or mRNA and plasmids that encode them," but the CMPs that were tested in the study could also be fused to Cas12a if needed for different applications. "There are many CRISPR systems available which enable multiplexing. CRISPR-chrom is compatible with many (if not all)."

 She also noted that MilliporeSigma's researchers haven't observed any increases in CRISPR off-targeting effects from the fusions of CMPs to Cas9. 

Now, it's up to customers to decide how they want to use the technology. "CRISPR-chrom can be implemented in the same way and for the same applications as any other CRISPR system. By genetically fusing CMP peptides to Cas9 or Cas12a, this enables compatibility with existing vectors and cellular delivery technology," Myers said. "We plan to combine CRISPR-chrom with additional technologies currently in development to offer next-generation, off-the-shelf CRISPR reagents, which provide unparalleled editing results from research through clinical applications. We will also apply these technologies in our Cell Design Studio custom cell-line engineering service group to expand our full-service capabilities accordingly."

She also noted that the company is continuing to watch the CRISPR field for high-impact developments in the areas of base editing, prime editing, and single-cell applications, and that MilliporeSigma will continue to research and develop new gene editing systems. 

"Ultimately, our goal is to provide the best-in-class reagent and service ecosystem from research through clinical applications," Myers added.

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