Multiplex genome engineering using CRISPR/Cas systems

Multiplex genome engineering using CRISPR/Cas systems. clone a uAb with desired specificity by replacing the existing DBP, namely -galactosidase-specific scFv13-R4, with a new target-specific DBP. All uAbs should be cloned into either a mammalian or bacterial expression vector, depending on the characterization route desired (Figure 2). First, identify a DBP that specifically binds LTX-401 the desired protein target. We have achieved success with a number of different formats including scFv intrabodies, FN3s, and DARPins. Other scaffolds may also work, provided that they are small, fold efficiently in the reducing cytosolic environment, and exhibit high affinity (low M to nM) and specificity towards the intended target (Sha, Salzman, Gupta, & Koide, 2017). There have been several reports describing successful fusions between DBPs and truncated E3 ubiquitin ligases (Caussinus et al., 2011; Portnoff et al., 2014; Shin et al., 2015). In our hands, the flexibility and solubility of human CHIP are advantageous properties in the context of uAbs; hence, these protocols will focus on the implementation of CHIP-based uAbs. The human E3 ligase CHIP (C-terminal U-box ligase domain) is modular in nature, containing an N-terminal tetratricopeptide repeat (TPR) domain, a helical linker domain, and a C-terminal U-box ligase domain (Figure 3a). The TPR domain binds molecular chaperones Hsc70-Hsp70 and Hsp90, resulting in the ubiquitination of a broad range of chaperone-bound Rabbit Polyclonal to OR10AG1 client proteins (Cyr, Hohfeld, & Patterson, 2002). The helical linker domain is necessary for protein dimerization and substrate ubiquitination (Nikolay et al., 2004). The U-box domain binds the E2 enzyme, facilitating the transfer of ubiquitin from the E2-ubiquitin complex to the substrate protein (Jiang et al., 2001). The uAbs described here utilize a truncated variant of CHIP, CHIPTPR, which lacks the substrate-recognition TPR domain (Figure 3a). Open in a separate window Figure 3 Design of genetic fusions for plasmid-based uAb expression(a) Linear representation of CHIP, CHIPTPR and scFv13-R4-based uAb (R4-uAb) that is specific for -galactosidase. Numbers refer to amino acid positions from N terminus (N) to C terminus (C). The proteins are aligned vertically with the coiled-coil and U-box domains of CHIP. CHIPTPR is a truncated version of CHIP lacking the TPR domain. R4-uAb was designed with an additional Gly-Ser LTX-401 (GS) linker connecting the scFv13-R4 intrabody to CHIPTPR. (b) Plasmid map for pcDNA3-R4-uAb, which encodes the R4-uAb in the mammalian expression vector, pcDNA3.1. A detailed description of how this plasmid was created can be found elsewhere (Portnoff et al., 2014). We employ a dual FLAG-6His tag on the C-terminus of our uAbs. LTX-401 The FLAG epitope (DYKDDDDK) aids in solubilization, while the 6His tag is used for purification. Both can be utilized for immunoblotting detection, although the FLAG-tag has a lysine residue, which can be ubiquitinated and potentially inaccessible to binding by the anti-FLAG antibody. Materials Plasmid DNA encoding uAb (pET28a-R4-uAb or pcDNA3-R4-uAb (Figure 3b); both described elsewhere (Portnoff et al., 2014) and available on Addgene #101800 & #101801) DBP DNA template Primers, with designed restriction overhangs DNA polymerase dNTPs Restriction enzymes Agarose (molecular biology quality) SybrSafe (Thermo Fisher Scientific) DNA gel extraction kit DNA ligase and buffer Competent cells (chemically or electro-competent) Antibiotics LTX-401 (based on expression vector chosen) Super Optimal Broth (SOB; see recipe) Luria-Bertani Agar (LBA; see recipe) Plasmid DNA miniprep kit Equipment Thermocycler Gel imaging system capable of detecting DNA Gel electrophoresis system Power supply Protocol.