(A) The experimental design. We designed a CRISPR-sgRNA library, in which each sgRNA targets specific tRNA gene family. Overall, we targeted nine proliferation-tRNAs, 10 differentiation- tRNAs and one pseudo tRNA family. Following cloning of the sgRNAs into a lenti-plasmid, we produced a lenti-viral pool that contained the entire sgRNA pool. Then, we transduced human cell lines (HeLa, WI38 fast and WI38 slow) expressing an inducible Cas9 with the lenti-viral sgRNA pool. We performed a CRISPR-edited tRNA cell competition by induction of the iCas9 in parallel to antibiotic selection (two biological repeats for each cell line). The iCas9 induction continued for 14 days, during which we sampled the heterogonous population every 3–4 days. Lastly, we deep-sequenced the sgRNAs in each sample, to evaluate the growth dynamics of the targeted-tRNA cells. (B) A heat map representation of the relative fitness of CRISPR-targeted tRNA variants in HeLa cells. Each row represents a CRISPR-targeted tRNA variant, colored according to the tRNA classification (proliferation-tRNAs in red, differentiation-tRNAs in blue and pseudo tRNA in black). Each column represents a time point during the iCas9 induction. The color code depicts a proxy of each row’s relative fitness - fold-change (log2) of the sgRNA read frequency in each time point relative to the sgRNA read frequency in day 0 of the iCas9 induction (see Materials and methods). The values were averaged over two biological repeats. (C) A scatter plot of the correlation between the expression of tRNAs in WT HeLa cells and their relative fitness upon CRISPR-targeting (regression model, relative fitness as a linear function of tRNA expression, had a slope of −219.5 and intercept of 0.26; Pearson correlation, r = −0.71, p<10−3). Expression levels for each targeted tRNA families is summed over all isodecoder genes of the family and is averaged over two biological repeats. Observed relative fitness of the CRISPR-targeted tRNA cells is shown given frequency of each tRNA family in day 7 of the competition. The colors and numbers denote the tRNA group. (D) Shown here is the estimated real relative fitness (log2) of each tRNA-targeted HeLa cells based on the observed relative fitness (log2), using a linear equation system. The observed and estimated real essentiality are correlated (regression model, estimated real relative fitness as a linear function of observed relative fitness, had a slope of 1.19 and intercept of 0.37; Pearson correlation, r = 0.93, p<10−5). The color code depicts the fraction of isodecoder genes of each ON-target tRNA family with full complementarity to the respective sgRNA. tRNA numbering in sub figures C and D is identical to those presented in sub figure B.