{Reference Type}: Journal Article
{Title}: Engineered FnCas12a with enhanced activity through directional evolution in human cells.
{Author}: Liu X;Liu X;Zhou C;Lv J;He X;Liu Y;Xie H;Wang B;Lv X;Tang L;Li M;Liu C;Zhao J;Liu Y;Song Z;Gu F;
{Journal}: J Biol Chem
{Volume}: 296
{Issue}: 0
{Year}: Jan-Jun 2021
暂无{DOI}: 10.1016/j.jbc.2021.100394
{Abstract}: Clustered regularly interspaced short palindromic repeat-Cas12a has been harnessed to manipulate the human genome; however, low cleavage efficiency and stringent protospacer adjacent motif hinder the use of Cas12a-based therapy and applications. Here, we have described a directional evolving and screening system in human cells to identify novel FnCas12a variants with high activity. By using this system, we identified IV-79 (enhanced activity FnCas12a, eaFnCas12a), which possessed higher DNA cleavage activity than WT FnCas12a. Furthermore, to widen the target selection spectrum, eaFnCas12a was engineered through site-directed mutagenesis. eaFnCas12a and one engineered variant (eaFnCas12a-RR), used for correcting human RS1 mutation responsible for X-linked retinoschisis, had a 3.28- to 4.04-fold improved activity compared with WT. Collectively, eaFnCas12a and its engineered variants can be used for genome-editing applications that requires high activity.