To obtain loss-of-function mutants, we designed two independent target sites at the first and second exon of LOC_Os10g34360, respectively (Fig. 4c and h).
To obtain loss of function mutants, we designed the target site at the third exon of 08SG2/OsBAK1 (Fig. 3a), then generated the CRISPR/Cas9 plasmid construct using the Biogle CRISPR/Cas kit (BGK03, http://www.biogle.cn/) according to instructions.
To generate the vector pET9c-mutOspA that expresses the OspA mutant (mutOspA), we designed primers to amplify the B. burgdorferi ospA gene lacking the signal sequence and we cloned the PCR fragment into pET9c.
To avoid possible phenotypic variation between different mutant alleles, we designed our analysis using cells from different individuals who each carried the identical, homozygous mutations within their NPC1 loci.
Indeed, most of the mutant chimeras that we designed (mutants 1 9; Figure 2C) contain all the Ca2+ ligands at the top loops and the two arginines at the bottom face of the C2B domain, and yet they are unable to rescue neurotransmitter release in synaptotagmin-1 KO neurons.
Most of the intron mutants that we designed have segment substitutions around 20 30 nt long.
To examine the mutant system thoroughly, we designed experiments where SNAP25 and mutant syntaxin-1 were separately expressed.
In order to discovery potent new mutant IDH1 inhibitors, we designed, synthesized and evaluated a series of allosteric mIDH1 inhibitors harboring the scaffold of 3-pyrazine-2-yl-oxazolidin-2-ones.
Using the W325F mutant as a template, we designed two additional mutants, F146W and S229W, at the binding sites on the thumb and palm subdomains, respectively, and one mutant, Y271W, near the active site on the finger subdomain.
Then, we designed mutants of RUNX2 with enhanced (cc+ mutants) or reduced (cc− mutants) CC propensity by using the same type of mutations that were able to effectively enhance or impair CC formation in RUNX2-ccd and in other CC peptides in the CD experiments (Fig. 2).
