1. Assembling of the CRISPR/Cas13a RNP complex (SYSTEM 1) v1

Abstract

Cas13a from Leptotrichia wadeii (LwaCas13a) will be used for all experiments in this proposal. The LwaCas13a protein is not currently available commercially, but the LwaCas13a protein vector for expression can be found on Addgene (pC013-Twinstrep-SUMO-huLwCas13a, Plasmid #90097) in the Feng Zhang Lab Plasmids section (Gootenberg et al., 2017). A previously published production and purification protocols for LwaCas13a will be followed according to Gootenberg et al., (2017) with some modifications described by Iwasaki and Batey (2020). The pET28-APho plasmid is a vector constructed in our laboratory. The plasmids will be transformed into BL21(DE3) Rosetta Escherichia coli cells. The functional activity of LwaCas13a and APhoproteins will be assessed by a reliable fluorescence signal to detect the activity when Cas13a starts to exert its collateral RNAse activity. The restriction map of the pC013 plasmid and pET28-APho plasmid are shown in the following Figure : For the assembling of the RNP complex, Synthego recommends sgRNA:Cas9 ratios between 3:1 and 9:1 for RNP formation in trasnfection experiments. However, the SHERLOCK system recommends a sgRNA:Cas13a ratio of 1:1. We will start with the ratio recommended by SHERLOCK.