Investigating Strategies to Increase Knock In Efficiency in Human Embryonic Stem Cells Through CRISPR Protein Engineering

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Cas9 proteins derive from both archaea and bacteria as a defense mechanism to protect against bacteriophage infection. Recently, these Cas9 proteins have been repurposed into powerful genetic engineering tools. However, the efficiency and specificity of wild-type Cas9 proteins need improvement to truly be implemented into medical treatment safely and effectively. I aimed to increase the editing efficiency of SpyCas9 (a programmable protein from the bacteria S. pyogenes that can induce breaks in DNA) and created a novel fusion protein termed MiCECas9 from two previous proteins (MiCas9 and CECas9) that had been shown to enhance editing efficiency on their own. These variants were inserted into cancer cell lines in an attempt to optimize editing procedures to then effectively edit human embryonic stem cells. By targeting two genes, LMNB1 (codes for the structural protein Lamin B in the nuclear lamina) and SEC61B (encodes the Endoplasmic Reticulum membrane transport protein Sec61 beta), I tested the efficiency of Homology Directed Repair in gene knock-in experiments. My study shows that while MiCECas9 did not improve editing efficiency, CECas9 shows great promise for increasing knock-in rates.