Pore-Based RNA Evaluation for Control of Integrity, Sequence, and Errors - Quality Control (PRECISE-QC)

Abstract

RNA is at the forefront of therapeutics and gene editing technologies. Yet, RNA synthesis remains expensive and low-yield. Consequently, most oligo manufacturers abstain from synthesizing RNA oligos longer than 60-mers. Solid-phase synthesis is the current standard production method but is often fraught with low coupling yields for canonical nucleotides and even poorer coupling for modifications. This results in high levels of byproducts such as truncations and RNA infidelity. Existing analytical methods can only provide quality control metrics such as RNA length distribution or limited composition information for short oligos. Here, we developed a standard quality control metric using Oxford Nanopore direct RNA sequencing to obtain direct insight into RNA length distribution, sequence, and presence of RNA modification sites. Our pipeline identifies error-prone regions and truncation sites that occur during synthesis. Furthermore, problematic steps in the synthesis are identified and repaired. We show that our platform can produce and assess CRISPR guide RNAs with high-fidelity and higher cleavage activity, and further, that modifications can be reliably detected. We envision that our tool will serve as an integral method for quality control pipelines that assess the integrity and accuracy of synthetic RNAs and guide the improved synthesis and yield of synthesized RNAs.

Keywords: Nanopore Sequencing; Quality Control; RNA Modifications; RNA synthesis; Solid phase synthesis.

Figure 1.

Overview of RNA solid-phase synthesis and quality control methods (A) Schematic of RNA solid-phase synthesis steps for extending RNA one base at a time starting from the initial 3’ nucleotide on the CPG bead. (B) List of error types and causes that can occur during solid-phase synthesis. (C) Plot of theoretical yield vs. RNA length based on the effects of coupling efficiency. (D) A comparison chart of quality control tools, showcasing the benefits of ONT DRS. (E) The suggested pipeline on how to integrate ONT DRS as a standard quality control for RNA synthesis.

Figure 2.

RNA synthesis sample preparation and analysis of truncated byproducts. (A) CRISPR-Cas9 system with sequence relevant regions of a single-guide RNA (sgRNA) highlighted. (B) RNA sample preparation schematic of ligating a 5’ adapter to the synthetic RNA to allow for full coverage sequencing of synthetic RNA crude sample using ONT DRS. (C) Computational pipeline to isolate adapter containing reads for further analysis. (D) Length distributions of first synthesis unmodified GFP sgRNA crude sample. (E) IGV and coverage plots of length subsets (<40 nt, 40 – 60 nt, and 60 – 90 nt) from first synthesis crude sample. Each gray bar on an IGV plot correlates to a single read. Rows that show a break in the gray bar equates to multiple reads placed on the same row. Peaks in the coverage show that many reads align to that particular region in the reference.

Figure 3.

Full-length RNA error analysis. (A) Error profile of in silico purified sgRNA before and after quality consultation. (B) Plot of number of errors that occur per full-length read. (C) in vitro CRISPR cleavage assay comparing percent of cleavage that occurs to eGFP-N1 linearized plasmid after 2 hours with sgRNA from first synthesis vs. optimized synthesis. Negative control is CRISPR reaction without sgRNA.

Figure 4.

Error rates by base type. (A) Schematic of which error is counted during error rate analysis. If a misincorporated base occurs after a correct base, it is counted in the error rates during analysis. (B) Bar plots of types of errors that occur by base type. (C) Confusion matrices of deletion and mismatch rates that occur by base type.

Figure 5.

Modification detection using ONT DRS. (A) Schematic of current levels caused by the 9-mer sequence that is present within the 9-mer space during translocation through the nanopore. (B) IGV plot showing m⁶A and Ψ detection using Dorado modification base calling. (C) Current signals of 9-mers containing Ψ at different positions compared to the unmodified sequence (top) as well as 9-mers containing m⁶A (bottom).