CRISPR/Cas9 genome editing

A pX330-based plasmid expressing Venus fluorescent protein (Shurtleff et al., 2016) was used to clone the gRNAs targeting Rab27a and Rab35. Two CRIPSR guide RNAs targeting each gene were selected using the CRISPR design tool (Hsu et al., 2013). For Rab27a, gRNAs targeting exon 4 and exon five were selected. For Rab35 both gRNAs targeted exon 1. Both gRNAs targeting each gene were cloned simultaneously in the pX330-Venus plasmid according to the PrecisionX Multiplex gRNA Kit instructions (SBI). MDA-MB-231 cells at 25% confluency were transfected for 48 hr, and then, they were trypsinized and sorted for single Venus-positive cells in a 96 well plate using a BD Influx cell sorter. Wells containing clones were allowed to expand (30 clones for Rab27a and 25 for Rab35) and knockouts for each gene were confirmed by immunoblots.