Fig. 4.

Highly efficient gene disruption mediated by Cas12a+. (A) Cas12a+ is a point mutant variant of LbCas12a harboring a D156R mutation. This variant is based on earlier work describing rationally designed variants of AsCas12a and has been shown to have increased activity in Arabidopsis (30, 31). (B) Scheme of the experimental workflow of the experiment shown in C. Gene-editing efficiency was measured by counting the number of nonfunctional alleles induced in the germline by testing for genetic complementation with known null alleles. Note that this method does not account for the induction of functional alleles. Levels of gene editing were in excellent agreement with levels measured by Amp-seq using the same crRNAs (Fig. 1D). (C) Cas12a+ induces much higher levels of gene editing than LbCas12a at all three target sites and at all tested temperatures. Dots represent the result of individual crosses of each condition and boxplots represent the 25% and 75% percentile and the median as a solid line. (D) Schematic of the experimental set-up of the experiment shown in E. Gene targeting by SpCas9 was performed using sgRNA transgenes of the Heidelberg CRISPR Fly Design library, which encode two sgRNAs expressed from the Gal4/UAS system (8). LbCas12a or Cas12a+ gene editing was induced with crRNA arrays encoding three crRNAs per gene encoded in pCFD8. Flies ubiquitously expressing either nuclease and the respective crRNA or sgRNAs were raised at 29 °C, 25 °C, or 18 °C and lethality was scored 5 d after the first flies eclosed. (E) Cas12a+ outperforms Cas9 and LbCas12a in identifying essential genes. Summary of the observed phenotypes is shown, with genes grouped according to the phenotype expected based on existing literature (noted below each group; see Materials and Methods). Semilethal refers to lethality with incomplete penetrance, where between 30% and 70% of animals do not survive. Two independent Cas9 sgRNA lines targeting wit and Pdk1 were available and each is represented by a small square. Targeting Raf with LbCas12a at 25 °C resulted in only female offspring, which likely reflects the fact that males have only a single copy of Raf, as it is located on the X chromosome. Differential mutagenesis of genes with limited prior knowledge has been analyzed in greater detail in SI Appendix, Fig. S9.