Difficulty in Measuring Gene Expression

In part characterization, one important measurement is the level of expression of a protein in vivo as it directly affects the activities of the constructs that are able to be captured. It is always a fine balance between disrupting the normal cell vitality and having sufficient expression to produce clear and resolved signals. The conventional approach to measure gene expression is through quantitative PCR, where cDNA of the mRNA transcripts are amplified to indicate expression level. However, due to the fundamental fluctuations of expression level and cell activities across different cells, seeking a house-keeping gene is often necessary to provide a common basis of measurement. While 

 Cas9-Fusion Proteins

One of the most exciting applications of Cas9-fusion proteins is in base editing. Fusion of RNA editing APOBEC1 cytidine deaminase, which can also perform DNA editing, allowed for site programmable C to T editing (Komor et al. 2016). Fusion of an evolved TadA Adenine deaminase allowed for A to G editing (Gaudelli et al. 2017). Unlike in HDR directed gene editing, base editors used a nickase cas9 (nCas9) that do not produce a double-strand break (DSB). Thus, it has comparatively higher editing frequency, with much lower undesired mutations such as indels. Base editors may represent a safer method for gene therapy, at least with regards to diseases treatable with single nucleotide changes possible with the current toolset.