An undergraduate laboratory class using CRISPR/Cas9 technology to mutate drosophila genes

Abstract

CRISPR/Cas9 genome editing technology is used in the manipulation of genome sequences and gene expression. Because of the ease and rapidity with which genes can be mutated using CRISPR/Cas9, we sought to determine if a single-semester undergraduate class could be successfully taught, wherein students isolate mutants for specific genes using CRISPR/Cas9. Six students were each assigned a single Drosophila gene, for which no mutants currently exist. Each student designed and created plasmids to encode single guide RNAs that target their selected gene; injected the plasmids into Cas9-expressing embryos, in order to delete the selected gene; carried out a three-generation cross to test for germline transmission of a mutated allele and generate a stable stock of the mutant; and characterized the mutant alleles by PCR and sequencing. Three genes out of six were successfully mutated. Pre- and post- survey evaluations of the students in the class revealed that student attitudes towards their research competencies increased, although the changes were not statistically significant. We conclude that it is feasible to develop a laboratory genome editing class, to provide effective laboratory training to undergraduate students, and to generate mutant lines for use by the broader scientific community. © 2016 by The International Union of Biochemistry and Molecular Biology, 44:263-275, 2016.

Keywords: CRISPR; drosophila; laboratory class; research; undergraduate.

Figure 1

Crossing scheme to identify CRISPR-induced mutants.

Figure 2. Design and analysis of mutations in TpnC4

A: GBrowse image from FlyBase.org showing the TpnC4 locus and annotated transcripts. Note that the gene is transcribed from right to left. The two sites targeted for mutation and the PCR primers used to analyze the mutant sequence are indicated below the annotated transcripts. B: Results of flight testing for four putative Deficiency/Mutant lines. Flies were released inside a plastic box and scored for their ability to fly Upwards (U), Horizontally (H), Downwards (D) or Not-at-all (N). Two lines (F2-1 and F9-8) showed normal flight, while two other lines (F2-3 and F9-5) were completely flightless. C: Analysis of TpnC4 protein accumulation in putative Deficiency/Mutant lines. Whole thoraces were homogenized in sample buffer and the constituent proteins were separated by SDS-PAGE alongside molecular weight standards. Separated proteins were transferred to nitrocellulose membrane, and stained with Ponceau S to visualize total protein (left panel, lanes 1–4). Note that there was equivalent loading between different samples. The blots were then incubated with an antibody that recognizes TpnC isoforms (right panel, lanes 6–9). Note that the control lines showing normal flight ability (F2-1 and F9-8) accumulated both TpnC4 and TpnC41C, whereas the flightless mutant lines (F2-3 and F9-5) only showed accumulation of TpnC41C. The absence of TpnC4 on the western blots corresponded to the inability of the lines to fly. D: Sequences of wild-type (WT) and mutant TpnC4 alleles in the region of the 5′ target. The vertical pipes ( | ) indicate the location of the 5′ and 3′ splice sites of exon 2, and the underlined sequence in WT denote the protospacer target sequence. In red: the two mutant alleles arise from small deletion (F2-3) or insertion (F9-5) of DNA, causing a shift in the reading frames and premature stop codons.

Figure 3. Design and analysis of mutations in Act57B

A: GBrowse image from FlyBase.org showing the Act57B locus and annotated transcript. The two sites targeted for mutation and the PCR primers used to analyze the mutant sequence are indicated below the annotated transcripts. B: Sequences of wild-type (WT) and mutant Act57B alleles in the region of the 5′ target. The underlined sequence in WT denotes the protospacer target sequence. The two mutant alleles arise from small deletions of DNA, causing a shift in the reading frames and identical TGA premature stop codons following the Gly codon.

Figure 4. Design and analysis of mutations in Act79B

A: GBrowse image from FlyBase.org showing the Act79B locus and annotated transcripts. The two sites targeted for mutation and the PCR primers used to analyze the mutant sequence are indicated below the annotated transcripts. Below that are diagrams indicating the regions deleted in the F1-5 and F3-3 alleles. B: Graph showing jumping ability of lines tested. Horizontal line indicates the jumping distance averaged over all tested genotypes. NT, not tested and data not included in calculation of average. C: Graph showing relative viability of the putative Deficiency/Mutant offspring as a percentage of all flies eclosed from each cross. Horizontal line indicates the viability averaged over all crosses analyzed. Lines that showed jumping ability and viability that were both reduced compared to the overall average were selected for PCR analysis. D: PCR analysis of two Deficiency/Mutant lines (F1-5 and F3-3) that showed reduced jumping and viability, alongside a control Deficiency/Mutant line (F8-9) where jumping ability was not reduced compared to the average. The wild-type PCR product is 2.8kb and was observed for the control genotype. PCR products of ~400bp and ~600bp were observed for F1-5 and F3-3 samples, respectively.