Abstract

CRISPR-based Functional Genomics (CFG) is a SciLifeLab core facility offering pooled CRISPR/Cas9 screening. The CRISPRCas9 system allows targeted gene knockout, editing, or transcriptional modulation. One powerful application of this technology is in pooled screening, where thousands of genes can be targeted in parallel in a population of cells. This population is then enriched for a phenotype of interest, typically by growth advantage, drug selection, or cell sorting. Finally, enriched and depleted guide RNAs are determined by Next Generation Sequencing (NGS).

All CRISPR guide libraries at CFG include a unique molecular identifier (UMI). This allows massively parallel lineage tracing and lineage dropout screening with improved reproducibility, precision and accuracy. UMIs reduce the number of cells needed to reach a set statistical power, or allow a more robust screen using the same number of cells. 

CFG supports pooled CRISPR/Cas9 screening from planning to data analysis. In addition to CRISPR knockout screening, which allows loss of function queries of coding genes, CFG now offers CRISPRi(nhibition) and a(ctivation), in which nuclease-dead Cas9 is fused to transcriptional effectors and targeted to elements of interest. CRISPRi is particularly useful for targeting ncRNAs and for work in primary cells, while CRISPRa also allows for gain-of- function screens. We are also implementing CRISPR/Cas9 screening coupled to single cell transcriptomics or to in situ sequencing and imaging. Finally, we are developing a novel method to direct saturating mutagenesis to elements of interest using the CRISPR system.

Karolinska Genome Engineering (KGE) is the precision editing branch of CRISPR Center Sweden. KGE offers a range of applications such as development of reporter cell lines (fluorescent proteins, luciferase, tags, etc.) and establishment of disease relevant cellular model systems such as knock-outs, knock-ins, creation/reversion of pathogenic mutations and cellular model systems to facilitate genetic screening and drug screening. It offers validation of hits from genetic screens or drug screens and in collaboration the Karolinska Center for Transgene Technologies, provides a seamless service pipeline from strategy design to transgenic animals. KGE also works with the iPS Core at Karolinska to generate edited iPS cell lines. KGE is open to novel applications and aims to meet any project needs and requirements.