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CRISPR technology has revolutionized life sciences research and drug development. Still a young technology albeit incredibly powerful, ongoing advances are providing better tools to increase the pace of research. Programming CRISPR for different target sites is achieved by changing the RNA guide sequence, therefore high purity, long guide RNA synthesis, and easy customization are key for achieving high performance and faster time to results. The ability to add chemical modifications at the right locations in guide RNAs can further enhance CRISPR performance.

Experts in RNA structure and function agree that predicting what modifications in a given RNA will be compatible with functional performance is im-possible without empirical testing. Agilent researchers were able to use their RNA synthesis chemistry, which excels at making longer RNA oligos, such as single guide RNAs (sgRNA), to study and elucidate the impact of different modifications on guide RNA performance.

Upgrading Stability and Specificity

RNA is very susceptible to degradation by enzymatic and chemical means both outside and inside cells. According to Laurakay Bruhn, PhD, Section Manager, Biological Chemistry, in Agilent Research Labs, early studies at Agilent identified successful strategies for adding modifications at the ends of guide RNAs to increase their resistance to exonucleases while maintaining function.

One version developed at Agilent called 3xMS, containing three 2’-O-methyl, 3’phosphorothioate modified nucleotides at both ends, has become a popular option for increasing activity including in more difficult to edit cells like primary and stem cells (see Figure).

Stability and activity are greatly enhanced by Agilent developed chemical modifications
Figure. Chemically modified sgRNAs facilitate high frequencies of indels and homologous recombination in cells. 2′-O-methyl (M), 2′-O-methyl 3′phosphorothioate (MS), 2′-O-methyl 3′thioPACE (MSP). Hendel et al, Nat Biotech 2015.

To explore how to improve the specificity of CRISPR using modified sgRNAs, Agilent performed systematic studies employing modifications in the 20-nt guide sequences of sgRNAs targeting four different clinically relevant genes and identified specific positions where modifications are particularly effective at improving specificity across a variety of guide sequences while maintaining high on-target activity as shown in Ryan et al. NAR 2018.

From Research through GMP

Many customers involved in preclinical and clinical studies using CRISPR technology are interested in high-quality sgRNAs from a single source that supplies research through GMP-grade materials.  With decades of investment spent improving the chemical synthesis of RNA and DNA, Agilent’s expertise in methods for purification and analysis of oligonucleotides is critical for ensuring high quality final products.

In addition to providing research-grade sgRNAs, Agilent has been manufacturing GMP oligonucleotide pharmaceuticals for over a decade. The high-purity sgRNAs, along with chemical modifications, and research-to-GMP synthesis capabilities, provide customers with the highest quality sgRNA, regardless of where they are in development, explains Dr. Bruhn.

Looking Forward

Agilent continues to develop new chemical modification strategies to improve sgRNA performance and offer customers insight on the right modifications for their applications. Agilent’s chemistry uniquely allows chemical synthesis of RNA lengths up to 160 nucleotides and longer. This capability enables exploration of applications for long RNA oligos including, for example, CRISPR-based gene activation and inhibition strategies that involve adding sequences to sgRNAs to impart new functionalities.

Agilent’s research- and GMP-grade chemically synthesized sgRNA adds to a growing portfolio of products for CRISPR including the SureGuide pre-designed and custom DNA oligo libraries for CRISPR-based screens to understand gene function, identify drug targets, and other applications using knockouts, CRISPRa, and CRISPRi libraries.

CRISPR technology is bringing an unprecedented power to virtually all areas of life sciences research and development of novel therapeutics. Agilent’s focus on world-leading quality in their DNA and RNA oligo products, in terms of fidelity, purity, and length, translates to savings for customers.

For DNA oligo libraries, increased library quality means screening fewer cells, and faster time to results. For chemically synthesized sgRNAs, high quality and purity combined with chemical modifications targeted to increase activity and specificity means customers can spend less time troubleshooting potential issues with guide RNA performance and more time achieving their research goals.

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