ena-STUDY-KAROLINSKA INSTITUTET-10-05-2019-04:06:59:197-5
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.
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.