Abstract
Delivering
CRISPR reagents (Cas9 and gRNA) can be difficult in many cell types, including
complex and relevant disease models. In the case of arrayed CRISPR screening,
we have the additional challenges of needing high knockout efficacies in
high-throughput. For example, a full-genome CRISPR screen requires ~20,000 gene
knockouts to be performed in separate wells of 384-well plates, with >70-85%
knockout efficiency required (depending on the assay endpoint). Unfortunately
commercial transfection reagents or electroporation are not always
effective or cost-efficient enough to use for a large arrayed screen, and these
limitations can delay or even prevent us from performing a screen at all. One
possible alternative is to use lipid nanoparticles (LNPs) to deliver Cas9 mRNA
and gRNA. LNPs contain mixtures of lipids, which can be optimised to transfect
many cell types which don’t respond to commercially-available reagents.
In AZ
Functional Genomics, we have implemented a high-throughput protocol to make and
dose thousands of different LNPs per day in a 384-well format. The small
reagent volumes and quick formulation time allows us to do large parallel and
iterative screening to optimize LNPs for CRISPR delivery in vitro. This
workflow enables rapid and cost-effective testing of transfection efficacy in
complex cell models, comparing hundreds of different LNP formulations and
Cas9/gRNA conditions. To perform a CRISPR screen, the most effective LNP lipid
formulation is loaded with Cas9 mRNA and gRNA to achieve an arrayed CRISPR
knockout.
Our LNP
formulation capability allows us to work on projects where the cell types are
not amenable to other transfection methods, where a screen cannot otherwise go
ahead.
