A novel chemo-enzymatic approach to making synthetic 100-unit oligonucleotide single guide RNA (sgRNA) increases both purity and yield, according to its developers at F. Hoffmann-La Roche and Genentech.
Writing in a recent paper, authors Filippo Sladojevich, PhD, senior principal scientist at Roche, and Stefan Koenig, PhD, distinguished scientist at Genentech, report consistent purity improvements ranging from 10 to 15% and increases in yield of three- to four-fold. Their method “leverages ligase-mediated assembly of two RNA fragments, each synthesized using standard solid-phase chemistry,” and “has been applied, without modification, to produce a variety of GMP-grade sgRNAs for our clinical ex vivo cell therapy pipeline.”
The process uses natural, non-engineered T4 RNA ligase 2 to synthesize the RNA fragments into the sgRNA targets, they note. “We used a combination of 49-mer variable fragments unique to each sgRNA, a single, shared 5’-phosphorylated 51-mer fragment, and a 29-mer DNA adaptor common to all sgRNAs. This combination brought together the RNA fragments to enable ligation,” Sladojevich and Koenig tell GEN.
For successful assembly using this process, Koenig and Sladojevich stress that buffer preparation is critical. Their team used an aqueous buffer comprised of hydroxymethyl aminomethane (Tris-HCI), magnesium chloride (MgCl2), dithiothreitol (DTT), and adenosine 5′-triphosphate (ATP) at pH 7.2.
This approach successfully produced multiple different sgRNAs, including sgRNA-412, sgRNA-401, sgRNA-930, sgRNA-467, and sgRNA-463, each of which can be used in CRISPR-Cas systems for gene editing or regulation.
When comparing HPLC purity between sgRNA’s produced using this process and vendor-sourced varieties, analysis showed that the sgRNA-412 developed by the team was 85.55 Area(A)% pure, while the sgRNA purchased from Vendor A was only at 47.26 A%, and the one from Vendor B at 71.12 A%. That trend applied to sgRNA-401 as well, with the newer method showing 88.21A% purity, compared to the commercial version from Vendor A at only 46.18 A%, and the version from Vendor B at 69.47 A% HPLC purity. For the remaining three sgRNAs (sgRNA-930, sgRNA-467, and sgRNA-463) developed by this new chemo-enzymatic method, purity was reported at 79.47 A%, 84.45 A%, and 84.55 A%, respectively.
After ligation, they said, “downstream processing included a single chromatographic purification and standard ultrafiltration steps, followed by isolation via lyophilization.
“The sgRNAs produced via the process are considered superior to longmers synthesized exclusively on solid-phase,” say Sladojevich and Koenig. “These in-house materials are being utilized for cell therapy manufacturing via CRISPR-Cas gene editing, and the disclosed process is considered readily usable for other investigative work in the field.”
The post Chemo-Enzymatic Method for Making sgRNA Triples Yield and Improves Purity appeared first on GEN - Genetic Engineering and Biotechnology News.
Writing in a recent paper, authors Filippo Sladojevich, PhD, senior principal scientist at Roche, and Stefan Koenig, PhD, distinguished scientist at Genentech, report consistent purity improvements ranging from 10 to 15% and increases in yield of three- to four-fold. Their method “leverages ligase-mediated assembly of two RNA fragments, each synthesized using standard solid-phase chemistry,” and “has been applied, without modification, to produce a variety of GMP-grade sgRNAs for our clinical ex vivo cell therapy pipeline.”
The process uses natural, non-engineered T4 RNA ligase 2 to synthesize the RNA fragments into the sgRNA targets, they note. “We used a combination of 49-mer variable fragments unique to each sgRNA, a single, shared 5’-phosphorylated 51-mer fragment, and a 29-mer DNA adaptor common to all sgRNAs. This combination brought together the RNA fragments to enable ligation,” Sladojevich and Koenig tell GEN.
For successful assembly using this process, Koenig and Sladojevich stress that buffer preparation is critical. Their team used an aqueous buffer comprised of hydroxymethyl aminomethane (Tris-HCI), magnesium chloride (MgCl2), dithiothreitol (DTT), and adenosine 5′-triphosphate (ATP) at pH 7.2.
This approach successfully produced multiple different sgRNAs, including sgRNA-412, sgRNA-401, sgRNA-930, sgRNA-467, and sgRNA-463, each of which can be used in CRISPR-Cas systems for gene editing or regulation.
When comparing HPLC purity between sgRNA’s produced using this process and vendor-sourced varieties, analysis showed that the sgRNA-412 developed by the team was 85.55 Area(A)% pure, while the sgRNA purchased from Vendor A was only at 47.26 A%, and the one from Vendor B at 71.12 A%. That trend applied to sgRNA-401 as well, with the newer method showing 88.21A% purity, compared to the commercial version from Vendor A at only 46.18 A%, and the version from Vendor B at 69.47 A% HPLC purity. For the remaining three sgRNAs (sgRNA-930, sgRNA-467, and sgRNA-463) developed by this new chemo-enzymatic method, purity was reported at 79.47 A%, 84.45 A%, and 84.55 A%, respectively.
After ligation, they said, “downstream processing included a single chromatographic purification and standard ultrafiltration steps, followed by isolation via lyophilization.
“The sgRNAs produced via the process are considered superior to longmers synthesized exclusively on solid-phase,” say Sladojevich and Koenig. “These in-house materials are being utilized for cell therapy manufacturing via CRISPR-Cas gene editing, and the disclosed process is considered readily usable for other investigative work in the field.”
The post Chemo-Enzymatic Method for Making sgRNA Triples Yield and Improves Purity appeared first on GEN - Genetic Engineering and Biotechnology News.