To prove that these motifs are solely responsible for the low knockout efficiency, we removed all of the other variables (e.g., locus context) by targeting sgRNA target sequences into the Lamin B1 (Lmnb1) locus of a Cas9-expressing mouse tumor cell line and expressing the respective sgRNA...
A rice genome sequence containing a synthesized target of presumed self-edited sgRNA (mTarget) was inserted into the SpCas9-NG-eBE3 vector. The same fragment was also inserted into SpCas9-eBE3 as a control. Arrows indicate the primers used to detect possible self-editing of sgRNA and the ...
BMC Bioinformatics (2020) 21:223 https://doi.org/10.1186/s12859-020-3531-9 METHODOLOGY ARTICLE Open Access CRISPRpred(SEQ): a sequence-based method for sgRNA on target activity prediction using traditional machine learning Ali Haisam Muhammad Rafid1,2†, Md. Toufikuzzaman1†, Mohammad ...
To assess the efficacy of this method, we attempted to knockin a sequence change in the recombinase-mediated cassette (loxPandloxP2272) into theRosa26locus using dual sgRNAs. To this end, we adopted a ssODN-mediated CRISPR/Cas9 KI strategy13,14, where we microinjected two sgRNAs sharing 16...
Consequently, we investigated the structural and sequence features of the core hairpin (the first stem loop of sgRNA) of SpCas9 sgRNA. We showed that the core hairpin structure of sgRNA is essential for SpCas9/sgRNA-mediated DNA cleavage and that the internal loop structure in the core hairpin...