Multiplexed conditional genome editing with Cas12a in Drosophila

Abstract

CRISPR-Cas genome engineering has revolutionized biomedical research by enabling targeted genome modification with unprecedented ease. In the popular model organism Drosophila melanogaster, gene editing has so far relied exclusively on the prototypical CRISPR nuclease Cas9. Additional CRISPR systems could expand the genomic target space, offer additional modes of regulation, and enable the independent manipulation of genes in different cells of the same animal. Here we describe a platform for efficient Cas12a gene editing in Drosophila We show that Cas12a from Lachnospiraceae bacterium, but not Acidaminococcus spec., can mediate robust gene editing in vivo. In combination with most CRISPR RNAs (crRNAs), LbCas12a activity is high at 29 °C, but low at 18 °C, enabling modulation of gene editing by temperature. LbCas12a can directly utilize compact crRNA arrays that are substantially easier to construct than Cas9 single-guide RNA arrays, facilitating multiplex genome engineering. Furthermore, we show that conditional expression of LbCas12a is sufficient to mediate tightly controlled gene editing in a variety of tissues, allowing detailed analysis of gene function in a multicellular organism. We also test a variant of LbCas12a with a D156R point mutation and show that it has substantially higher activity and outperforms a state-of-the-art Cas9 system in identifying essential genes. Cas12a gene editing expands the genome-engineering toolbox in Drosophila and will be a powerful method for the functional annotation of the genome. This work also presents a fully genetically encoded Cas12a system in an animal, laying out principles for the development of similar systems in other genetically tractable organisms for multiplexed conditional genome engineering.

Keywords: CRISPR; Cas12a; Cas9; Drosophila; genome engineering.

Fig. 1.

LbCas12a mediates efficient temperature-sensitive gene editing in Drosophila. (A) Schematic of the Cas12a genome engineering system. Cas12a possesses RNase activity and can cleave crRNA arrays into individual crRNAs. Upon on-target binding, Cas12a cuts dsDNA, producing DSBs with 5′ overhangs. These are often imprecisely repaired by the endogenous DNA repair machinery, resulting in mutations at the target locus. (B) Schematic of the workflow of experiments presented in C and D. Single Cas12a crRNAs are cloned into expression vectors pCFD7 (for AsCas12a) or pCFD8 (for LbCas12a). Plasmids are integrated at defined genomic locations and crossed to the respective nuclease. Offspring inheriting Cas12a and crRNA transgenes are raised at different temperatures and assessed for on-target gene editing. (C) Cas12a-mediated mutagenesis of ebony (e) at different temperatures. Images of flies expressing crRNA-e and either AsCas12a or LbCas12a and raised at the indicated temperatures are shown. Disruption of e results in dark coloration of the cuticle. AsCas12a mediates efficient mutagenesis only at 29 °C, while LbCas12a mediates gene disruption at all tested temperatures. (D) Assessment of gene-editing efficiency of 11 target sites by Amp-seq. Activity of each nuclease was tested at 18 °C, 25 °C, and 29 °C. Genomic target sites of each crRNA were PCR amplified and amplicon pools were subjected to deep sequencing. Gene-editing activity was strongly temperature-sensitive with both nucleases. LbCas12 displayed much more robust activity, mediating strong mutagenesis with 7 of 11 crRNAs at 29 °C, compared to 2 of 11 in the case of AsCas12a. Rates of modified reads being called due to sequencing errors was between 0.5% and 1.6% (SI Appendix, Fig. S1).

Fig. 2.

Multiplexed gene editing through crRNA arrays. (A) Gene editing of evi with three arrayed crRNAs. crRNAs were expressed from a U6:3 promoter and combined with nos-Gal4 UAS-LbCas12a transgenes for germline-restricted mutagenesis. Flies were raised at 29 °C and viable, indicative of efficient restriction of gene disruption to the germline. nos-Gal4 UAS-LbCas12a pCFD8-evi^3x males were crossed to wild-type virgin females and genomic DNA from individual offspring was subjected to PCR amplification of the evi locus. PCR amplicons were sequenced and sequencing traces were parsed into wildtype and alternative allele sequences (Materials and Methods). The majority of animals harbor mutant evi alleles modified at the target sites of crRNA1 and crRNA2. Edited sequences are indicated in red and the crRNA target site is indicated in green, the PAM in yellow, and the window in which LbCas12a is expected to cut is shaded in gray. Indels at individual target sites are typically larger than 10 bp, further supporting data presented in SI Appendix, Fig. S4. (B) Gene editing of y with three arrayed crRNAs. The experiment was conducted as described above using a pCFD8-y^3x transgene. Sequencing of PCR amplicons revealed highly efficient gene editing at the target site of the third crRNA in the array and editing with low efficiency at the target site of the first crRNA. No mutations were detected at the target site of the second crRNA. (C) Due to the compact size of Cas12a, several crRNAs can be encoded on commercially available oligonucleotides and oligos can be fused to construct larger crRNA arrays encoded in pCFD8 (see SI Appendix, Supplementary Cloning Protocol). (D) Multiplex gene targeting of four genes with two crRNAs each. Flies transgenic for a 8x crRNA array and act5C-LbCas12a were raised at 29 °C and had ebony cuticle and bristles displaying the singed phenotype. Each target locus was PCR amplified and amplicons were subjected to Sanger sequencing. Efficiency of gene editing was inferred from sequencing traces by ICE analysis (Materials and Methods). High levels of activity were detected for crRNA1 targeting e, crRNAs 7 and 8 had intermediate activity and crRNAs 5 and 6 showed low to medium activity. crRNAs 2 to 4 were inactive. crRNAs 1 (crRNA-e in Fig. 1 C and D), 2 (crRNA-e6) and 4 (crRNA-se) were in parallel also tested when expressed as single crRNAs in pCFD8 (Fig. 1D) with consistent results.

Fig. 3.

Tissue-specific gene editing with UAS-LbCas12a. (A) LbCas12a-mediated mutagenesis of e in the pnr-Gal4 expression domain along the dorsal midline leads to the development of dark pigment in the cuticle. Flies were raised at 29 °C. (B) Mutagenesis of evi in the posterior compartment of the wing imaginal disc with hh-Gal4. Evi protein is detected throughout the wing imaginal disc in control and hh-Gal4 UAS-LbCas12a U6:3-crRNA-evi^3x animals raised at 18 °C, but absent from the posterior compartment in animals raised at 29 °C, demonstrating efficient, temperature-sensitive, and spatially defined gene disruption. (Scale bars, 50 μm.) (C) Mutagenesis of ct in the posterior compartment with hh-Gal4. Ct protein is detected in a stripe of cells in control and hh-Gal4 UAS-LbCas12a U6:3-crRNA-evi^3x animals raised at 18 °C, but absent from the posterior compartment in animals raised at 29 °C, demonstrating efficient, temperature-controlled and spatially defined gene disruption. (Scale bars, 50 μm.) (D) Tissue-specific mutagenesis of essential genes in the wing primordium with nub-Gal4. Wings from animals expressing nub-Gal4 UAS-LbCas12a and the indicated crRNA array are shown. When combined with ubiquitous act5C-LbCas12a these crRNAs lead to lethality, but tissue-restricted expression with nub-Gal4 gives rise to viable or semiviable flies. Wings of such animals display specific and informative phenotypes affecting wing size, vein formation, bristle formation, or wing margin formation. (E) Intestinal tumorigenesis induced by conditional LbCas12a mutagenesis of N. esg-Gal4 was used to express LbCas12a in adult intestinal stem cells. esg-Gal4 UAS-LbCas12a U6:3-crRNA-N^3x animals raised at 29 °C are lethal before adulthood, consistent with the known expression of esg-Gal4 in larval and pupal tissues. Reducing the culture temperature to 18 °C leads to viable adults that develop intestinal tumors characterized by expression of pros, a hallmark of tumors caused by loss of N, indicating low level mutagenesis at this temperature. Mutagenesis of y was used as a control, as y does not play a role in intestinal stem cells. (Scale bars, 50 μm.) (F) Efficient Cas12a-mediated mutagenesis in the germline to create heritable alleles. Images in A–E are representative examples of at least nine samples from two independent experiments.

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.