The following protocol was used to generate the CLiP2 mutants: the Chlamydomonas CC-5415 strain was grown in TAP medium in a 500mL flask under 100 μmol photons m-2 s-1 light to a density of 0.5-2 × 10^6 cells/mL. Cells were collected by centrifugation at 1000 x g for 5 min in batches of 50 mL. Pellets were washed twice with 2.5 mL GeneArt® MAX Efficiency® Transformation Reagent for Algae (Thermo Fisher Scientific), and then resuspended in GeneArt® MAX Efficiency® Transformation Reagent for Algae at 2-3 x 10^8 cells/mL. 50 μL cell suspensions were then aliquoted in pre-chilled 1.5 mL tubes. For each tube, 5ng of CIB2 DNA at 5 ng/μL was added to the cell suspension and mixed by tapping the tube. The mixture was then transferred to a pre-chilled 2mm electroporation cuvette (12358-346, Bulldog- Bio). Electroporation was immediately performed with a NEPA21 electroporator (Nepa Gene) with the following settings: poring pulse: voltage: 300V; pulse length: 8 msec; pulse interval: 50 msec; number of pulses: 2; decay rate: 40%; polarity: +; transfer pulse: voltage: 20V; pulse length: 50 msec; pulse interval: 50 msec; number of pulses: 5; decay rate: 40%; polarity: +/-. For ~20% of the transformations reactions, the poring pulse decay rate was set to 10%, instead of 40%, and the transfer pulse number of pulses was set to 1, instead of 5. After electroporation, the cells in the cuvette were incubated at room temperature for 15 min and then transferred to a 15mL tube containing 8 mL TAP supplemented with 40 mM sucrose and shaken gently in the dark for 6 h. After incubation, cells were collected by centrifugation at 1000 x g for 5 min and resuspended in 1 mL TAP. Cells were then plated (250 μL per plate) on rectangular PlusPlatesTM (Singer Instruments) containing ~70mL TAP agar with 20 μg/mL hygromycin and incubated in low light ((<5 μmol photons m-2 s-1 light) for approximately two weeks before colony picking. Colonies were then robotically picked and arrayed at 384 colonies per plate as previously described.