New Drosophila Circadian Clock Mutants Affecting Temperature Compensation Induced by Targeted Mutagenesis of Timeless

Drosophila melanogaster has served as an excellent genetic model to decipher the
molecular basis of the circadian clock. Two key proteins, PERIOD (PER) and TIMELESS
(TIM), are particularly well explored and a number of various arrhythmic, slow, and fast
clock mutants have been identified in classical genetic screens. Interestingly, the free
running period (tau, t) is influenced by temperature in some of these mutants, whereas
t is temperature-independent in other mutant lines as in wild-type flies. This, so-called
“temperature compensation” ability is compromised in the mutant timeless allele “ritsu”
(tim rit), and, as we show here, also in the tim blind allele, mapping to the same region of
TIM. To test if this region of TIM is indeed important for temperature compensation, we
generated a collection of new mutants and mapped functional protein domains involved
in the regulation of t and in general clock function. We developed a protocol for targeted
mutagenesis of specific gene regions utilizing the CRISPR/Cas9 technology, followed by
behavioral screening. In this pilot study, we identified 20 new timeless mutant alleles with
various impairments of temperature compensation. Molecular characterization revealed
that the mutations included short in-frame insertions, deletions, or substitutions of a
few amino acids resulting from the non-homologous end joining repair process. Our
protocol is a fast and cost-efficient systematic approach for functional analysis of
protein-coding genes and promoter analysis in vivo. Interestingly, several mutations with
a strong temperature