CRISPR-Mediated Knockout of FLOT1 and AGA

Five different mammalian Cas9 genome editing constructs encoding gRNAs for FLOT1 were obtained from Horizon Discovery (Cambridge, UK). The vector backbone for all constructs was pD1301-AD. Guide RNA sequences are shown in Table 1.
For knocking out the expression of aspartylglucosaminidase (AGA) in HEK293T cells, gRNAs were designed with the “optimized CRISPR design” tool (www.crisp.mit.edu) and cloned into PX459 (Addgene #48139) [2 (link)]. Transfection and cultivation of single-cell clones was performed as described for FLOT1. For the screening of single-cell clones, AGA enzyme activity was measured as described [11 (link)]. The gRNA sequence used was 5’-CACCGATACCCTCCAATAATTTGGC-3’.