Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors

Abstract

CRISPR interference (CRISPRi) enables programmable, reversible, and titratable repression of gene expression (knockdown) in mammalian cells. Initial CRISPRi-mediated genetic screens have showcased the potential to address basic questions in cell biology, genetics, and biotechnology, but wider deployment of CRISPRi screening has been constrained by the large size of single guide RNA (sgRNA) libraries and challenges in generating cell models with consistent CRISPRi-mediated knockdown. Here, we present next-generation CRISPRi sgRNA libraries and effector expression constructs that enable strong and consistent knockdown across mammalian cell models. First, we combine empirical sgRNA selection with a dual-sgRNA library design to generate an ultra-compact (1-3 elements per gene), highly active CRISPRi sgRNA library. Next, we compare CRISPRi effectors to show that the recently published Zim3-dCas9 provides an excellent balance between strong on-target knockdown and minimal nonspecific effects on cell growth or the transcriptome. Finally, we engineer a suite of cell lines with stable expression of Zim3-dCas9 and robust on-target knockdown. Our results and publicly available reagents establish best practices for CRISPRi genetic screening.

Generation of Schwann cell derived melanocytes from hPSCs identifies pro-metastatic factors in melanoma

“…For this purpose, we used the MeWo melanoma cell line, which is also significantly enriched for a T2 melanocyte transcriptional signature (Normalized enrichment score = 1.25, adjusted p-value = 0.0018). We used a previously established CRISPR interference (CRISPRi) system 42 for targeted gene knockdowns, employing a pool of guide RNAs (gRNAs) targeting the 100 genes that make up the T2 signature, as well as the 100 T1 genes as a control set. We transduced MeWo cells that constitutively express dCas9-KRAB and luciferase with the gRNA-expressing lentiviruses in a pooled fashion and injected them into the tail veins of NSG (NOD scid gamma) mice for lung colonization assays.…”

“…A dCas9-KRAB-mCherry lentiviral construct (pHR-UCOE-EF1A-dCas9-HA-2xNLS-XTEN80-KRAB-P2A-mCherry) 42 was used to generate CRISPRi-competent MeWo and A375 cells. HEK293T cells were maintained in DMEM supplemented with 10% FBS, penicillin, streptomycin and amphotericin B, and passaged using 0.05% Trypsin prior to cells reaching 90% confluency.…”

Targeting epigenetic aberrations of sarcoma in CRISPR era

“…201 Large-scale CRISPRi screening was successfully performed using Zim3-dCas9 and dual sgRNA libraries. 202 The study identified AARS, DNAJC19, and POLR1D in K562 leukemia cells. Screening for altered CREs in acute leukemia followed by CRISPR/dCas9-p300 or CRISPR/dCas9-KRAB identified oncogenic and tumor-suppressive CREs.…”

“…The screening of CREs at the ARID5B locus identified variants that affect transcription factor binding; in addition, heritable acute lymphoblastic leukemia risk variants were identified 201 . Large‐scale CRISPRi screening was successfully performed using Zim3‐dCas9 and dual sgRNA libraries 202 . The study identified AARS, DNAJC19 , and POLR1D in K562 leukemia cells.…”

In vivo perturb-seq of cancer and microenvironment cells dissects oncologic drivers and radiotherapy responses in glioblastoma

“…1A). Following tumor establishment, lentiviral dual-sgRNA libraries 15 targeting 48 genes that were nominated from in vitro CRISPRi growth or radiation modifier screens (Supplementary Fig. 1 and Supplementary Table 1) plus non-targeting sgRNA controls were delivered to intracranial tumors using CED.…”