An efficient and precise method for generating knockout cell lines based on CRISPR-Cas9 system.

Although the efficiency and versatility of CRISPR-Cas9 system has been greatly improved over conventional genome editing methods such as zinc finger or TALEN, it is still time-consuming and labor-intensive for screening knockout/knock-in cell clones due to differences of the targeted location or efficacies of guide RNAs (gRNAs). Here, we adapted a targeted knock-in strategy with CRISPR-Cas9 system and characterized the efficiency for generating single or double knockout cell lines. Specifically, a homology-arm based donor cassette consisting of genes encoding a fluorescence protein and antibiotic selection marker driven by a constitutive promoter was co-transfected with a gRNA expressing unit. Based on FACS sorting and antibiotic drug selection, positive cell clones were confirmed by genotyping and at the protein expression level. The results indicated that more than 70% of analyzed clones identified by cell sorting and selection were successfully targeted in both single and double knockout experiments. The procedure takes less than three weeks to obtain knockout cell lines. We believe that this methodology could be applicable and versatile in generating knockout cell clones with high efficiency in most cell lines.

Structural insight into a matured humanized monoclonal antibody HuA21 against HER2-overexpressing cancer cells.

HER2, a member of the epidermal growth factor receptor (EGFR) family, has been associated with human breast, ovarian and gastric cancers. Anti-HER2 monoclonal antibodies (mAbs) have demonstrated clinical efficacy for HER2-overexpressing breast cancer. A chimeric antibody chA21 that specifically inhibits the growth of HER2-overexpressing cancer cells both in vitro and in vivo has previously been developed. To reduce a potential human anti-mouse immune response, the humanized antibody HuA21 was developed and was further subjected to affinity maturation by phage display on the basis of chA21. Here, the crystal structure of HuA21-scFv in complex with the extracellular domain of HER2 is reported, which demonstrates that HuA21 binds almost the same epitope as chA21 and also provides insight into how substitutions in HuA21 improve the binding affinity compared with chA21, which could facilitate structure-based optimization in the future. Furthermore, the effects of HuA21 variants with constant domains of different lengths were explored and it was noticed that the deletion of constant domain 1 could improve the inhibition efficacy in a cell-proliferation assay, possibly functioning via increased internalization, which might guide the design of other monoclonal antibodies.

Crystallization and preliminary X-ray diffraction analysis of Gos1p, a yeast SNARE protein.

The Gos1 protein (Golgi SNAP receptor complex member 1) is involved in the SNARE complex, which is the core machinery that drives membrane fusion between cargo-carrying vesicles and their target membranes in the secretory and endocytic pathways in yeast. Truncated versions of the Gos1 protein from Saccharomyces cerevisiae were cloned, expressed, purified and crystallized. The crystal belonged to space group P212121, with unit-cell parameters a=39.67, b=43.58, c=81.94 Å, α=ß=γ=90°. An X-ray diffraction data set was collected at 100 K to 1.63 Šresolution. Matthews coefficient (VM) calculations suggest that one molecule is present in the asymmetric unit, corresponding to a solvent content of ∼55%.