Methods to characterize enzymes for genome engineering

Providing a plurality of individual discrete samples comprising populations of cells, preferably mammalian cells, preferably human cells, wherein each population of cells overexpresses both (i) a single genome engineering protein or a variant thereof and (ii) a reporter protein, wherein (i) and (ii) are expressed in a known ratio, preferably 1:1, in the samples;

• lysing the cells to release the proteins;
• normalizing levels of the genome engineering proteins or variants thereof based on levels of the reporter protein;
• allowing the genome engineering proteins or variants thereof to combine with a guide RNA under conditions sufficient to form ribonucleoprotein complexes in each sample;
• contacting each sample with a plurality of analysis substrates, under conditions sufficient for the genome engineering protein or variant thereof to act on one or more of the substrates;
• determining levels of each of the analysis substrate in each sample at a plurality of times;
• and
• calculating rate of depletion or enrichment of each of the analysis substrates from each sample.