Sequence features associated with the cleavage efficiency of CRISPR/Cas9 system

Abstract

The CRISPR-Cas9 system has recently emerged as a versatile tool for biological and medical research. In this system, a single guide RNA (sgRNA) directs the endonuclease Cas9 to a targeted DNA sequence for site-specific manipulation. In addition to this targeting function, the sgRNA has also been shown to play a role in activating the endonuclease activity of Cas9. This dual function of the sgRNA likely underlies observations that different sgRNAs have varying on-target activities. Currently, our understanding of the relationship between sequence features of sgRNAs and their on-target cleavage efficiencies remains limited, largely due to difficulties in assessing the cleavage capacity of a large number of sgRNAs. In this study, we evaluated the cleavage activities of 218 sgRNAs using in vitro Surveyor assays. We found that nucleotides at both PAM-distal and PAM-proximal regions of the sgRNA are significantly correlated with on-target efficiency. Furthermore, we also demonstrated that the genomic context of the targeted DNA, the GC percentage, and the secondary structure of sgRNA are critical factors contributing to cleavage efficiency. In summary, our study reveals important parameters for the design of sgRNAs with high on-target efficiencies, especially in the context of high throughput applications.

CRISPR applications in ophthalmologic genome surgery

“…[54] Additionally, alterations in the PAM sequence from canonically acceptable sequences can ablate cutting ability. Courtney et al used this characteristic of sgRNA-SpCas9 complexes to specifically target the C395T mutation in the KRT12 gene, a dominant-negative allele for Meesmann epithelial corneal dysplasia (MECD) in heterozygous patients.…”

Bioinformatic analysis of Listeria monocytogenes CRISPR

“…The CRISPR structure has three major features: a set of Cas genes, an AT-rich leader sequence, and palindromic direct repeats separated by variable sequences called spacers [5]. The repeats are highly conserved, always contain palindromic motifs, and may constitute RNA secondary structure [6]. Unique spacer sequences are usually derived from mobile genetic elements such as plasmids and phages [7] while Cas genes are often adjacent to the CRISPR loci.…”

CRISPR/Cas9-mediated traceless gene correction and activation of the HBB locus in iPSCs with the beta thalassaemia mutation

“…Different sgRNAs can have different activities [146]. This is of relevance when a composite DNA break is needed.…”

“…Testing the applicability of the paired nickase strategy, which is predicted to have higher specificity compared to the native CRISPR-Cas9, for targeted gene correction of the HBB mutation is one of the main objectives of this thesis. Because the activity of the endonuclease (nickase or double nickase) and its induced HDR depends on different variables that include the targeted locus, gRNA sequence, and type of cells used [117,[145][146][147], it was expected that this strategy would require extensive optimisation.…”

“…The efficiency of DNA modifications (through NHEJ or HDR) in response to composite DNA breaks induced by paired nickase is affected by the sequence context, the offset distance between pairs of guides used to target the intended region, and the distance of the nicks relative to the intended modification [96, 145,146]. The intended DNA modifications in the current study involve a corrective substitution of the disease-causing mutation and an insertion of an excisable dual selection cassette using the piggyBac transposon-based approach described in Yusa et al [182].…”