Exploring the importance of sequencing guide RNAs in Functional Genomics.

Guide RNAs, or single-guide RNAs (sgRNAs), are indispensable tools in functional genomics and transcriptome modulation. Their programmable nature and their ability to direct Cas9 or other Cas-endonucleases to specific genomic loci underpins the utility of these systems in knockout studies, transcriptional activation/repression, RNA editing, and more (Wu et al, 2014; Kim et al, 2024). This blog reviews the main scenarios where sequencing of sgRNAs by NGS is relevant. It also provides suggestions on the appropriate strategies to prepare libraries to sequence sgRNAs, based on their structural features.

Stably expressed viral delivery systems

Verification of sgRNA sequences is critical whenever guides are delivered from lentiviral or adeno-associated viral (AAV) vectors, since these platforms support long-term co-expression of the nuclease and guide (Liu et al, 2017). Sequencing ensures that the intended guide is present, intact, and functional. This is important for experimental reproducibility. Below, we outline a few use cases where sgRNA sequencing provides important quality control and risk mitigation.

Plasmid-library quality control prior to packaging

Sequencing the plasmid pool before virus production confirms library complexity, uniform guide representation, and the absence of synthesis or cloning artifacts, providing a reference baseline for subsequent screen analysis (Zhou et al, 2023).

Pooled lentiviral screens

Genome-wide or focused CRISPR screens rely on high-complexity lentiviral libraries that integrate one sgRNA per cell. Post-selection sequencing of the integrated guide region is essential for deconvolving barcode counts, calculating enrichment/depletion statistics, and assigning gene-level phenotypes (Mathiowetz et al, 2023; Bock et al, 2022).

Single or low multiplex delivery

Even when only one or a few guides are packaged, sequencing of the expressed sgRNA is recommended to confirm functionality and rule out point mutations or recombination events introduced during cloning or reverse transcription, which could lead to confusion about functional read-outs or off-target assessments.

Ex vivo cell delivery

Ex vivo therapeutic approaches where patient-derived cells are modified (e.g., CAR-T, genome-edited T cells or modified stem-cells), require sequence verification of gRNAs prior to re-infusion. This ensures accurate on-target editing and avoids unintended batch variability. It is also a requisite for submissions and regulatory oversight from US Food Drug Administration, European Medicines Agency and the Pharmaceuticals and Medical Devices Agency (FDA, 2002; EMA 2018; PMDA 2019).

In vivo delivery

For in vivo gene editing, especially using AAV or non-integrating viral vectors, regulatory expectations are higher due to direct patient exposure and prolonged expression risks. Complete sequencing of the gRNA cassette ensures guide integrity, validates identity, and rules out deleterious insertions or recombination events (FDA, 2022; EMA 2018; PMDA 2019)

Manufacturing Control Applications

Control of guide identity

Synthetic sgRNA production demands rigorous identity verification. Sequencing confirms the nucleotide composition and the correct targeting region, eliminating ambiguity introduced by chemical synthesis errors, mislabelling, or oligonucleotide cross-contamination.

Control of guide purity

Each nucleotide addition during solid-phase RNA synthesis has a coupling efficiency below 100%. As the RNA length increases, the likelihood of producing full-length molecules declines, and truncated byproducts (e.g., N–1, N–2 species) accumulate. For RNAs of approximately 100 nucleotides, such as sgRNAs, the reduction in yield is already significant (Rees et al., 2021). Even with purification methods like HPLC or PAGE, it remains technically challenging to separate full-length sgRNAs from closely sized truncated products, especially for sequences prone to strong secondary structure or base-specific retention behaviors (Kanavarioti, 2019). The emergence of sequencing as a quality control step offers an orthogonal approach to assess molecular purity and detect undesired species.

Strategies for sgRNA sequencing

The length and structure of sgRNAs vary depending on the CRISPR-Cas system: Cas9 sgRNAs are typically ~100 nt, while Cas12 and Cas13 crRNAs are shorter, around 40–60 nt. In Cas9 systems, which are commonly used, the sgRNA consists of a 17–20 nucleotide spacer sequence that directs target recognition, and an ~80–100 nt tracrRNA-derived scaffold that enables Cas9 binding and activation (Jinek et al, 2012) (Figure 1). In contrast, Cas12a uses a ~42–44 nt sgRNA that combines a 20–24 nt spacer with an ~18–22 nt direct repeat-derived scaffold forming a hairpin (Zetsche et al, 2015). Cas13 systems, which target RNA rather than DNA, use sgRNAs composed of a ~24–30 nt spacer and a ~24 nt scaffold, and the Cas13 protein self-processes these guides to form an active ribonucleoprotein complex (Abudayyeh et al., 2017).