Benchmarking Perturbation Tools for the Noncoding Genome

Deciphering the functionality of the noncoding genome which includes important cis -regulatory elements (CREs) and transcribed noncoding RNA genes remains technically challenging. Here, using massively parallel genetic screening, we systematically benchmark the performance of five representative loss-of-function perturbation tools, including single guide RNA (gRNA) mediated SpCas9 cleavage or CRISPR interference, and paired gRNA (pgRNA) involved dual-SpCas9, Big Papi (paired SpCas9 and SaCas9) or dual-enAsCas12a fragment deletion methods, in decoding the roles of the noncoding genome. For targeting CREs such as enhancer, dual-SpCas9 outperforms other methods with superior efficiency of destroying functional genomic regions. For perturbing noncoding RNA genes, in addition to dual-SpCas9, other RNA-targeting methods such as RNA interference are recommended to discriminate transcript-dependent or -independent roles. A deep learning model DeepDC with associated web server is built to facilitate optimal dual-SpCas9 pgRNA design for efficiently deleting a genomic fragment. Together, our work provides practical guidance on selecting appropriate loss-of-function tools to resolve the functional complexity of the noncoding genome.