Parallel profiling of combinatorial mutations optimizes CRISPR's genome-editing performance

The combined effect of multiple mutations on protein function is hard to predict, thus the ability to functionally assess a vast number of protein sequence variants would be enormously useful for protein engineering. Here we present a high-throughput platform to enable scalable assembly and parallel characterization of barcoded protein variants with high-order combinatorial modifications. We illustrate this platform, CombiGEM via seamless ligation (CombiSEAL), by systematically profiling a library of 948 combination mutants of the widely used Streptococcus pyogenes Cas9 (SpCas9) nuclease to optimize its genome-editing activity in human cells. The ease of pool-assessing editing activities of SpCas9s at multiple on- and off- target sites accelerates the identification of optimized variants and facilitates the study of mutational epistasis. We successfully identify Opti-SpCas9 that possesses enhanced editing specificity without sacrificing potency and broad targeting range. This platform is broadly applicable for engineering any protein through experimenting combinatorial modifications en mass.