Biology subjects

Barrera, L. A.

Publications and source records attributed to Barrera, L. A..

2 recordsLinked to original sources

bioRxiv2018

Pairwise library screen systematically interrogates Staphylococcus aureus Cas9 specificity in human cells

We report a high-throughput screening approach to measure Staphylococcus aureus Cas9 (SaCas9) genome editing variation in human cells across a large repertoire of 88,692 single guide RNAs (sgRNAs) paired with matched or mismatched target sites in a synthetic cassette. We incorporated randomized barcodes that enable whitelisting of correctly synthesized molecules for further downstream analysis, in order to circumvent the limitation of oligonucleotide synthesis errors. We find SaCas9 sgRNAs with a 21-nucleotide spacer are most active against off-targets with single and double mismatches, compared to shorter or longer sgRNAs. Using this dataset, we developed an SaCas9 specificity model that performs well in ranking off-target sites. The barcoded pairwise library screen enabled high-fidelity recovery of guide-target relationships, providing a scalable framework for the investigation of CRISPR enzyme properties and general nucleic acid interactions.

bioengineering↗

bioRxiv2017

The experimental design and data interpretation in “Unexpected mutations after CRISPR–Cas9 editing in vivo” by Schaefer et al. are insufficient to support the conclusions drawn by the authors

To the Editor To the Editor Conflict of Interest Statement References References The recent correspondence to the Editor of Nature Methods by Schaefer et al.1 has garnered significant attention since its publication as a result of its strong conclusions contradicting numerous publications in the field using similar analytical approaches and methods2-4. The authors suggest that the CRISPR-Cas9 system is highly mutagenic in genomic regions not expected to be targeted by the gRNA. We believe that the conclusions drawn from this study are unsubstantiated by the disclosed experiments as they were designed and carried out. Further, it is impossible to ascribe the observed differences in the subject mice to the effects of CRISPR per se. The genetic differences seen in ...

genetics↗