gRNA靶点设计

CRISPR basics

The CRISPR/Cas9 gene editing system is used for a variety of genetic modifications, including gene knockout, knockin, activation, and inhibition, allowing for exploration of a range of applications from identification of gene function in cell and animal models to editing disease-causing genes in humans.

The CRISPR/Cas9 system contains two crucial components: the Cas9 endonuclease and guide RNA (gRNA). The gRNA, a short RNA sequence, directs Cas9 to the target site by binding to complementary DNA in the target genome. Cas9, an RNA-guided DNA endonuclease, introduces double-stranded breaks (DSBs) at specific genomic locations, as long as the target sequence is adjacent to a short DNA motif known as the Protospacer Adjacent Motif (PAM). Once DSBs are introduced at target sites, cells use either Non-Homologous End Joining (NHEJ), or Homology Directed Repair (HDR) pathway to correct the break (Figure 1).

While NHEJ may facilitate the creation of gene knockout, it lacks precision and produces a heterogeneous population of edited cells. HDR, on the other hand, uses a donor DNA template that allows for introduction of precise changes including directed point mutations and large fragment knockins and knockouts at the site of the DSB.

Figure 1. Mechanisms of CRISPR/Cas9-induced DNA repair.

Designing gRNAs

A gRNA consists of two main parts: the guide sequence and the scaffold sequence. The guide is a variable region that is complementary to the target DNA next to a PAM site. The scaffold is a constant region of about 80 nucleotides that forms a stem-loop structure necessary for Cas9 binding, as shown in Figure 2. For most CRISPR applications, the guide and scaffold are fused into a single guide RNA (sgRNA) for experimental simplicity. For successful CRISPR applications, the guide sequence should be long enough to ensure specificity but minimize off-target effects; typically, about 20 nucleotides long, with a GC content of 40–60%.

Figure 2. The structure of sgRNA with spCas9 specific scaffold sequence.

When designing gRNAs, it is important to ensure high on-target efficiency and to minimize the risk of off-target effects. For most applications, including simple gene knockouts, a single gRNA paired with the appropriate Cas9 is sufficient to produce the desired phenotype. In some cases, however, dual gRNAs are a better choice. For example, using Cas9(D10A) nickase with two offset gRNAs targeting opposite strands at the same site can create double-strand breaks, which in turn reduces off-target effects (Figure 3). Dual gRNAs are also used to delete a DNA fragment located between two DSBs induced by the gRNA pair or to target two different genes simultaneously. The gRNA must target a region within close proximity of the PAM sequence, which depends on the selected Cas9 variant; hence, it is important to ensure gRNA compatibility with the specific Cas9 variant being used. Additionally, secondary structures within the gRNA should be minimized, as they can potentially make the guide sequence unavailable for pairing with the target DNA, thereby reducing efficiency.

You can find more information about optimizing the different components of the CRISPR system here, and information about different delivery methods for CRISPR components here.

Figure 3. Nickase activity with two gRNAs.