Abstract
BackgroundThe CRISPR/Cas9 system can effectively introduce site-specific modifications to the genome. The efficiency is high enough to induce targeted genome modifications during embryogenesis, thus increasing the efficiency of producing genetically modified animal models and having potential clinical applications as an assisted reproductive technology. Because most of the CRISPR/Cas9 systems introduce site-specific double-stranded breaks (DSBs) to induce site-specific modifications, a major concern is its potential off-targeting activity, which may hinder the application of the technology in clinics. In this study, we investigated off-targeting events in genome edited pigs/fetuses that were generated through direct injection of the CRISPR/Cas9 system into developing embryos; off-targeting activity of four different sgRNAs targeting RAG2, IL2RG, SCD5, and Ig Heavy chain were examined.ResultsFirst, bioinformatics analysis was applied to identify 27 potential off-targeting genes from the sgRNAs. Then, PCR amplification followed by sequencing analysis was used to verify the presence of off-targeting events. Off-targeting events were only identified from the sgRNA used to disrupt Ig Heavy chain in pigs; frequency of off-targeting was 80 and 70% on AR and RBFOX1 locus respectively. A potential PAM sequence was present in both of the off-targeting genes adjacent to probable sgRNA binding sites. Mismatches against sgRNA were present only on the 5′ side of AR, suggesting that off-targeting activities are systematic events. However, the mismatches on RBFOX1 were not limited to the 5′ side, indicating unpredictability of the events.ConclusionsThe prevalence of off-targeting is low via direct injection of CRISPR/Cas9 system into developing embryos, but the events cannot be accurately predicted. Off-targeting frequency of each CRISPR/Cas9 system should be deliberately assessed prior to its application in clinics.
Highlights
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system can effectively introduce site-specific modifications to the genome
We investigated off-targeting events in RAG2. Target gene (RAG2)/IL2RG. Target gene (IL2RG) double knockout pigs, SCD5. Target gene (SCD5) knockout fetuses, and Ig Heavy chain knockout pigs previously generated via direct injection of the CRISPR/Cas9 system
Most of the potential off-targeting loci were located in intron regions and only a portion of the potential off-targeting sites possessed –NGG sequences that can act as a protospacer adjacent motif (PAM) sequence (Additional file 1: Table S1-S4)
Summary
The CRISPR/Cas system can effectively introduce site-specific modifications to the genome. The efficiency is high enough to induce targeted genome modifications during embryogenesis, increasing the efficiency of producing genetically modified animal models and having potential clinical applications as an assisted reproductive technology. Because most of the CRISPR/Cas systems introduce site-specific double-stranded breaks (DSBs) to induce site-specific modifications, a major concern is its potential off-targeting activity, which may hinder the application of the technology in clinics. Because introducing site-specific double-stranded breaks (DSB) on the target locus is the foundation of many genome-editing tools [12], off-targeting events have been reported with the use of CRISPR/Cas system [13,14,15]. The frequency of off-targeting highly depends on the design of single guide RNA (sgRNA) [3], the complimentary RNA sequence that attracts Cas to the target genome locus
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