CRYPTOCHOME proteins are necessary for mammalian circadian rhythms and have many well-established biochemical roles within the molecular clock. While studies examining the effect of null Cry alleles have been informative, they have failed to dissect out the relative importance of, and the molecular mechanisms behind, the many roles of the CRY1 and CRY2 proteins. To address this, we created an allelic series of Cry mutants through random mutagenesis, followed by a cell-based screen to isolate mutants with aberrant repression of CLOCK-BMAL1. We identified 22 mutants with mutations resulting in single amino acid substitutions which cause a variety of deficiencies in different CRY functions. To illustrate the breadth and value of these new tools, we present an in-depth analysis of two of these mutants, CRY2G354D and CRY2G351D; the former shows deficiency in clock protein binding and is required for repression by both CRYs, while in contrast, the latter displays normal binding function but exhibits a CRY2-specific repression phenotype. Further, while overexpression of CRY2 in NIH 3T3 cells caused a dose-dependent decrease in rhythm amplitude, overexpression of CRY2G351D abolished rhythmicity. In summary, characterization of these unique alleles provides new opportunities for more-sophisticated insight into the multifaceted functions of the CRY proteins in circadian rhythms.Organisms from cyanobacteria to humans have evolved circadian rhythms to anticipate the dramatic changes in environmental conditions that occur on a daily basis. The driving force behind the 24-h periodicity of these endogenous rhythms is a cell-autonomous molecular oscillator, composed of transcriptional/translational feedback loops (reviewed in reference 1).In mammals, the Cryptochrome (CRY) proteins CRY1 and CRY2 play an integral role in the circadian clock by closing the core negative-feedback loop (9,12,28,29). In 1999, two groups showed that mice lacking both Crys demonstrate a complete loss of rhythm in circadian wheel-running behavior when put in constant darkness (26,28), suggesting an essential role for murine CRY proteins (mCRYs) in the core molecular mechanism. This year, it was shown that both CRY1 and CRY2 repressed CLOCK-BMAL1-mediated transcription much more potently than the mammalian PERIOD (PER) proteins in transiently transfected cells (9, 12). While it is known that CRYs are essential for circadian rhythms at both the behavioral (28, 29) and molecular (21) levels, many questions concerning how the various known actions of the CRY proteins contribute to their essential roles in the molecular mechanism of the circadian clock remain unanswered.Mammalian CRY1 and CRY2 have several well-established biochemical roles in the molecular oscillator in addition to their role in repressing the CLOCK-BMAL1 heterodimer. Both CRY1 and CRY2 bind the PER proteins (9, 12), and this interaction affects PER's stability (30) and localization (12). While the CRY interaction domain on PER has been described, the regions important for PER binding on C...