Short talk:
Sequential genetic dependencies identified by time-resolved orthogonal gene editing

Ronay Cetin1, Martin Wegner1, Sarada Saud1, Leah Luwisch1, Simone Schaubeck1, Eva Quandt1,2, Rupert Ă–llinger3, Roland Rad3, Manuel Kaulich1,4,5

1Institute of Biochemistry II, Faculty of Medicine, Goethe University Frankfurt, Frankfurt am Main, Germany,                
2Faculty of Medicine and Health Sciences, Universitat Internacional de Catalunya, Barcelona, Spain,
3Institute of Molecular Oncology and Functional Genomics, TUM School of Medicine, Technical University of Munich, Munich, Germany,
4Frankfurt Cancer Institute, Frankfurt am Main, Germany,
5Cardio-Pulmonary Institute, Frankfurt am Main, Germany

CRISPR-based gene editing is a powerful tool for functional genomics that enables unbiased investigations of single and combinatorial genotype-to-phenotype associations. In the light of efforts to map all combinatorial gene dependencies in cancer, choosing a robust CRISPR system is a very important consideration. Cas9 and enCas12a have been used for combinatorial screening, however, a side-by-side comparison of these two enzymes in combinatorial CRISPR screens remains elusive. Moreover, genetic dependency screens have so far focused on the simultaneous perturbation of two genes of interest. This is, however, in contrast to the development of cancer or therapy resistance, in which mutations occur sequentially, with their order directing clonal evolution. In this work, we performed a side-by-side comparison of parallel and orthogonal combinatorial gene editing approaches and present optimized metrics for combinatorial genotype-phenotype associations. Our analyses identified Cas9 to yield higher phenotypic resolutions in single and combinatorial conditions when compared to enCas12a. This is likely explained by the overall slow establishment of enCas12a phenotypes, which we demonstrate to depend on the pre- crRNA processing activity of enCas12a. Interestingly, we reveal the orthogonal gene editing system CHyMErA to also suffer from Cas12a pre-crRNA processing activity and present an arrayed gRNA-expression system (CHyMErA.v3) that circumvents gRNA processing, enabling highly efficient orthogonal gene editing screens. Lastly, we applied our learned parameters and developed a time-resolved orthogonal gene editing system which we used to uncover the hitherto unexplored concept of sequential genetic dependencies.

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