Generalizable sgRNA design for improved CRISPR/Cas9 editing efficiency.
Ontology highlight
ABSTRACT: Motivation
The development of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has provided a simple yet powerful system for targeted genome editing. In recent years, this system has been widely used for various gene editing applications. The CRISPR editing efficacy is mainly dependent on the single guide RNA (sgRNA), which guides Cas9 for genome cleavage. While there have been multiple attempts at improving sgRNA design, there is a pressing need for greater sgRNA potency and generalizability across various experimental conditions.Results
We employed a unique plasmid library expressed in human cells to quantify the potency of thousands of CRISPR/Cas9 sgRNAs. Differential sequence and structural features among the most and least potent sgRNAs were then used to train a machine learning algorithm for assay design. Comparative analysis indicates that our new algorithm outperforms existing CRISPR/Cas9 sgRNA design tools.Availability and implementation
The new sgRNA design tool is freely accessible as a web application, http://crispr.wustl.edu.Supplementary information
Supplementary data are available at Bioinformatics online.
SUBMITTER: Hiranniramol K
PROVIDER: S-EPMC7203743 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The CRISPR/Cas9 gene editing system has enhanced the development of genetically engineered animals for use in xenotransplantation. Potential limitations to the CRISPR/Cas9 system impacting the development of genetically engineered cells and animals include the creation of off-target mutations. We sought to develop a method to reduce the likelihood of off-target mutation while maintaining a high efficiency rate of desired genetic mutations for the GGTA1 gene. Extension of sgRNA length, responsible for recognition of the target DNA sequence for Cas9 cleavage, resulted in improved specificity for the GGTA1 gene and less off-target DNA cleavage. Three PAM sites were selected within exon 1 of the porcine GGTA1 gene and ten sgRNA of variable lengths were designed across these three sites. The sgRNA was tested against synthetic double stranded DNA templates replicating both the native GGTA1 DNA template and the two most likely off-target binding sites in the porcine genome. Cleavage ability for native and off-target DNA was determined by in vitro cleavage assays. Resulting cleavage products were analyzed to determine the cleavage efficiency of the Cas9/sgRNA complex. Extension of sgRNA length did not have a statistical impact on the specificity of the Cas9/sgRNA complex for PAM1 and PAM2 sites. At the PAM3 site, however, an observed increase in specificity for native versus off-target templates was seen with increased sgRNA length. In addition, distance between PAM site and the start codon had a significant impact on cleavage efficiency and target specificity, regardless of sgRNA length. Although the in vitro assays showed off-target cleavage, Sanger sequencing revealed that no off-target mutations were found in GGTA1 knockout cell lines or piglet. These results demonstrate an optimized method for improvement of the CRIPSR/Cas9 gene editing system by reducing the likelihood of damaging off-target mutations in GGTA1 knocked out cells destined for xenotransplant donor production.
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Project description:The current commonly used single-guide RNA (sgRNA) structure has a shortened duplex compared with the native bacterial clustered regularly interspaced short palindromic repeats RNA (crRNA)–transactivating crRNA (tracrRNA) duplex. Here we show that modifying the sgRNA structure by extending the duplex length and mutating the fourth T of the continuous sequence of Ts (which is the pause signal for RNA polymerase III [pol III]) to C or G significantly, and sometimes dramatically, improves knockout efficiency in cells. In addition, the new sgRNA structure also significantly increases the efficiency of more challenging genome-editing procedures, such as gene deletion, which is important for inducing a loss-of-function in non-coding genes.
2015-11-17 | GSE74766 | GEO
Project description:BackgroundDue to post-cleavage residence of the Cas9-sgRNA complex at its target, Cas9-induced DNA double-strand breaks (DSBs) have to be exposed to engage DSB repair pathways. Target interaction of Cas9-sgRNA determines its target binding affinity and modulates its post-cleavage target residence duration and exposure of Cas9-induced DSBs. This exposure, via different mechanisms, may initiate variable DNA damage responses, influencing DSB repair pathway choices and contributing to mutational heterogeneity in genome editing. However, this regulation of DSB repair pathway choices is poorly understood.ResultsIn repair of Cas9-induced DSBs, repair pathway choices vary widely at different target sites and classical nonhomologous end joining (c-NHEJ) is not even engaged at some sites. In mouse embryonic stem cells, weakening the target interaction of Cas9-sgRNA promotes bias towards c-NHEJ and increases target dissociation and reduces target residence of Cas9-sgRNAs in vitro. As an important strategy for enhancing homology-directed repair, inactivation of c-NHEJ aggravates off-target activities of Cas9-sgRNA due to its weak interaction with off-target sites. By dislodging Cas9-sgRNA from its cleaved targets, DNA replication alters DSB end configurations and suppresses c-NHEJ in favor of other repair pathways, whereas transcription has little effect on c-NHEJ engagement. Dissociation of Cas9-sgRNA from its cleaved target by DNA replication may generate three-ended DSBs, resulting in palindromic fusion of sister chromatids, a potential source for CRISPR/Cas9-induced on-target chromosomal rearrangements.ConclusionsTarget residence of Cas9-sgRNA modulates DSB repair pathway choices likely through varying dissociation of Cas9-sgRNA from cleaved DNA, thus widening on-target and off-target mutational spectra in CRISPR/Cas9 genome editing.
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