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Genetic interaction mapping and exon-resolution functional genomics with a hybrid Cas9-Cas12a platform

Nat Biotechnol. 2020-03; 
Gonatopoulos-Pournatzis T, Aregger M, Brown KR, Farhangmehr S, Braunschweig U, Ward HN, Ha KCH, Weiss A, Billmann M, Durbic T, Myers CL, Blencowe BJ, Moffat J.
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Oligo pool The oligo pools were synthesized on 90k microarray chips by CustomArray Inc. (a member of GenScript), each with a density of ~94,000sequences. Oligos were amplified by PCR over ten cycles using Q5 polymerase ((1) 98°C, 30s; (2) 98°C, 10s; (3) 53°C, 30s; (4) 72°C, 10s; (5) 72°C, 2min; steps 2–4 repeated for nine cycles). Amplified oligos were purified on a PCR purification column, and an aliquot was run on 2% agarose gel to check purity. Get A Quote

摘要

Systematic mapping of genetic interactions (GIs) and interrogation of the functions of sizable genomic segments in mammalian cells represent important goals of biomedical research. To advance these goals, we present a CRISPR (clustered regularly interspaced short palindromic repeats)-based screening system for combinatorial genetic manipulation that employs coexpression of CRISPR-associated nucleases 9 and 12a (Cas9 and Cas12a) and machine-learning-optimized libraries of hybrid Cas9-Cas12a guide RNAs. This system, named Cas Hybrid for Multiplexed Editing and screening Applications (CHyMErA), outperforms genetic screens using Cas9 or Cas12a editing alone. Application of CHyMErA to the ablation of mammalian paral... More

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