Saturation mutagenesis1,2 – coupled to an appropriate biological assay – represents a fundamental means of achieving a high-resolution understanding of regulatory3 and protein-coding4 nucleic acid sequences of interest. However, mutagenized sequences introduced in trans on episomes or via random or “safe-harbor” integration fail to capture the native context of the endogenous chromosomal locus5. This shortcoming markedly limits the interpretability of the resulting measurements of mutational impact. Here, we couple CRISPR/Cas9 RNA-guided cleavage6 with multiplex homology-directed repair (HDR) using a complex library of donor templates to demonstrate saturation editing of genomic regions. In exon 18 of BRCA1, we replace a six base-pair (bp) genomic region with all possible hexamers, or the full exon with all possible single nucleotide variants (SNVs), and measure strong effects on transcript abundance attributable to nonsense-mediated decay and exonic splicing elements. We similarly perform saturation genome editing of a well-conserved coding region of an essential gene, DBR1, and measure relative effects on growth that correlate with functional impact. Measurement of the functional consequences of large numbers of mutations with saturation genome editing will potentially facilitate high-resolution functional dissection of both cis-regulatory elements and trans-acting factors, as well as the interpretation of variants of uncertain significance observed in clinical sequencing.

Massively parallel reporter assays (MPRAs) functionally screen thousands of sequences for regulatory activity in parallel. To date, there has been no systematic comparison of differences in MPRA design. Here, we screen a library of 2,440 candidate liver enhancers and controls for regulatory activity in HepG2 cells using nine different MPRA designs. We identify subtle but significant differences that correlate with epigenetic and sequence-level features, as well as differences in dynamic range and reproducibility. We also validate en masse that enhancer activity is robustly independent of orientation, at least for our library and designs. Finally, with a new method, we assemble and test the same enhancers as 192-mers, 354-mers, and 678-mers, and observe surprisingly large differences. This work provides a framework for the experimental design of high-throughput reporter assays, suggesting that the extended sequence context of tested elements, and to a lesser degree the precise assay, influence MPRA results.