Direct sequencing

Genotyping by direct sequencing.

The advantage of using a high-efficiency experimental setup, such as fully transgenic CRISPR/Cas, is that finding the desired mutants becomes increasingly easy. Using our cas9 fly lines and functional U6:3-gRNA transgenes (note that not all gRNAs work) we now consistently achieve germline transmission rates of 100% (i.e. every tested cas9; gRNA expressing animal gives rise to mutant offspring) and the number of total offspring which have mutations at the gRNA target site is usually >40%. Even when injecting donor constructs to make precise sequence changes the number of positive offspring is often >10%.

The high frequency of desired events makes it practical to identify flies harboring the desired sequence alteration by direct sequencing. In the first instance we typically genotype around 20 flies.

 

Step 1: Cross candidate flies

Cross flies expressing cas9 and gRNA transgenes to appropriate partners (e.g. a balancer or wild-type strain). Select candidate offspring (potentially heterozygous for the desired mutation) and cross single flies to appropriate partners (e.g. a balancer or wild-type strain). Use offspring from several cas9;gRNA parents. Once larva appear in the tubes collect the candidate flies in PCR tubes.

Step 2: Isolate genomic DNA

Isolate genomic DNA from candidate flies. We use Microlysis-Plus (Microzone, UK) according to the instructions in the manual, which requires minimal hands-on time. But there are multiple protocols for genomic DNA extraction available online (e.g. here).

Step 3: PCR the target locus

Run a PCR reaction to amplify the target locus. Fwd and rev primers that bind ~300bp 5′ and 3′ of the gRNA target site usually work well. 

Step 4: Purify PCR product

Sequencing will usually require to clean up the PCR reaction. We usually do a gel extraction, which ensures that no unspecific amplification products are carried over into the sequencing reaction. Other purification methods should also work.

Step 5: Send product for sequencing.

Send the PCR product for Sanger sequencing using one of the primers used for the PCR.

Step 6: Analyse sequencing trace

The sequencing trace will return an overlay of the sequence from both alleles. Mutations will produce double-peaks in the sequencing chromatogram (as will naturally occurring SNPs). The two bases at each position can be usually unambiguously read and since the wild-type sequence is known from the database the sequence of the mutated chromosome can be identified. An example trace from the ebony locus is shown below. (Note: In rare cases only one alleles will yield a PCR product. We had that case with a region of the wingless gene balanced over cyo. In that case sequencing only returned chromatograms with single peaks which originated from the mutagenized chromosome.)

 

sequChromatogram