Fig. 2: Multiplexing beyond 2 guides with Cas12a.

A 7mer arrays were constructed with all combinations of either an essential or non-essential guide at each position (2^7 = 128 species), using the same DR sequences at each position, in three independent sets with unique gRNA sequences targeting the same genes at each position (n = 384 total). B Guide sets were evenly represented in the combined pool before and after packaging and transduction (C) 7mer guide array representation is consistent across replicates and variation is consistent with high quality screens. D Fold change of guide arrays vs. number of essential guides on the array (n = 384 arrays). E Fold change vs. position of essential guide on array, for all arrays encoding one essential guide (six nonessentials). F Fold change of guide arrays encoding one essential per array, forward vs. reverse orientation. Essential guides expressed at positions 6 and 7 deviate from the diagonal, indicating position-specific loss of editing. G A linear regression model that learns single gene knockout effects can be used to predict combinatorial target phenotype, an accurate null model for genetic interactions.