For all screens, each plasmid library was transfected along with
plasmids provided with the ViraPower Lentiviral Expression into 293T cells. At
48 and 72h post transfection, supernatant were collected, filtered using a 40
μm steriflip filtration system (EMD Millipore). For arrayed experiments,
individual plasmids were transfected and viruses produced as described above.
For pHAGE-scKO and arrayed/pooled pLGB-scKO vector experiments, virus was
concentrated using Peg-it virus concentration solution (SBI). Viral titer of the
concentrated lentiviral library was determined by transduction of MCF10A-Cas9
cells for 48h at several viral dilutions, splitting cells into replica plates,
and subjecting replica plate to blasticidin. Percent control growth was used to
assess MOI. MCF10A-Cas9 cells with estimated MOIs of 0.3 carried forward.
For pHAGE-scKO and arrayed/pooled pLGB-scKO vector experiments, media
was switched to 1ug/mL doxycycline to induce expression of Cas9 in pCW-Cas9
cells. LentiCas9-Blast cells were used for CROP-seq experiments. Editing was
allowed take place for 14d for arrayed and pooled pLGB-scKO and 21d for
pHAGE-scKO and CROP-seq experiments. Media was changed every 48h and cells were
cultured every 96h. For the first half of editing, cells were cultured in the
presence of 5μg/mL blasticidin and 0.5μg/mL puromycin to ensure
high sgRNA and Cas9 expression.
plasmids provided with the ViraPower Lentiviral Expression into 293T cells. At
48 and 72h post transfection, supernatant were collected, filtered using a 40
μm steriflip filtration system (EMD Millipore). For arrayed experiments,
individual plasmids were transfected and viruses produced as described above.
For pHAGE-scKO and arrayed/pooled pLGB-scKO vector experiments, virus was
concentrated using Peg-it virus concentration solution (SBI). Viral titer of the
concentrated lentiviral library was determined by transduction of MCF10A-Cas9
cells for 48h at several viral dilutions, splitting cells into replica plates,
and subjecting replica plate to blasticidin. Percent control growth was used to
assess MOI. MCF10A-Cas9 cells with estimated MOIs of 0.3 carried forward.
For pHAGE-scKO and arrayed/pooled pLGB-scKO vector experiments, media
was switched to 1ug/mL doxycycline to induce expression of Cas9 in pCW-Cas9
cells. LentiCas9-Blast cells were used for CROP-seq experiments. Editing was
allowed take place for 14d for arrayed and pooled pLGB-scKO and 21d for
pHAGE-scKO and CROP-seq experiments. Media was changed every 48h and cells were
cultured every 96h. For the first half of editing, cells were cultured in the
presence of 5μg/mL blasticidin and 0.5μg/mL puromycin to ensure
high sgRNA and Cas9 expression.