Parallel CRISPR-Cas9 screens clarify impacts of p53 on screen performance

Bowden, A.; Morales-Juarez, David A; Sczaniecka-Clift, Matylda; Agudo, Maria Martin; Lukashchuk, Natalia; Thomas, John Christopher; Jackson, Stephen

doi:10.1101/2020.02.20.957746

“…Despite a computational method for the estimation of the gene-dependency level while taking account of the copy-number effect, it is experimentally demanding to determine copy numbers for cell types of interest, while it is still difficult to resolve mismatch-tolerance effect 11,16 . What's more, it has recently been reported that Cas9-induced DSBs posed obstacles to high-throughput screens in human non-transformed cells via p53-dependent cell growth arrest [17][18][19][20] . High-efficiency Cas9 editing could cause cell death in human pluripotent stem cells (hPSCs) 20 and G1 cell cycle arrest in hTERT RPE1 cells 18 .…”

Section: Introductionmentioning

confidence: 99%

Genome-wide interrogation of gene functions through base editor screens empowered by barcoded sgRNAs

Xu

¹

,

Liu

²

,

Liu

³

et al. 2021

View full text Add to dashboard Cite

Canonical CRISPR screens rely on Cas9-induced DNA double-strand breaks (DSBs) to generate targeted gene knockouts. These DSB-dependent methodologies may yield false-positive results by mistakenly assuming targeted loci as essential for cell viability, especially when high-copy-number sites are targeted. Here, we use CRISPR cytosine base editors for genome-scale knockout screens by perturbing gene start codons or splice sites, or by introducing premature termination codons (PTCs).Combining with iBAR strategy we have previously established, we realized an iBARed cytosine Base Editing-mediated gene KnockOut (BARBEKO) screening strategy at a genome-scale (targeting 17,501 genes) in multiple human cell lines. By constructing such a cell library through lentiviral infection at a high MOI (up to 10), we significantly reduced starting cells while producing screening results with improved efficiency and accuracy. More importantly, in comparison with Cas9-mediated cell fitness screens, BARBEKO screens are no longer affected by DNA-cleavage induced cytotoxicity in HeLa, K562, or DSB-sensitive RPE1 cells. We anticipate that BARBEKO offers a valuable tool to complement the current CRISPR screens in various settings.We aim to re-establish CRISPR loss-of-function screening strategy with the following beneficial features: allowing high MOI screening to improve efficiency and economy, ideal for both positive and negative screens, and applicable for screening in non-transformed cells such as primary cells, hPSCs, and RPE1 that usually carry p53 and are sensitive to DSB damage. The simple solution could be the combination of iBAR strategy and CRISPR base editor-mediated gene knockouts. CRISPR-STOP and iSTOP approaches have been proposed to utilize the CRISPR-based cytosine base editor 3 (BE3, C•G to T•A) to introduce nonsense mutations for gene silencing 24,25 . It is foreseeable to achieve broader coverage of genes using CBEs to include additional sites for sgRNA design, splice acceptor sites (Gapinske et al., 2018), splice donor sites and translation initiation sites.Here, we established a genome-wide iBARed cytosine Base Editing-mediated gene KnockOut (BARBEKO) screening strategy, in which cytosine base editors perturb genes by disrupting splicing sites or translation initiation sites, or introducing premature termination codons (PTCs), and all sgRNAs were re-designed to carry iBARs 23 . BARBEKO approach in genome-scale has been applied in multiple cell lines, HeLa, K562 and RPE1 cells, all at high MOIs for screens of cell fitness. We envision that BARBEKO strategy might be particularly useful for CRISPR screens in complex models such as primary cells, organoids and in vivo studies, where the source of cells is usually limited and sensitive to DNA damage, and it is hard, if not impossible, to control transduction efficiency in making libraries.

show abstract

“…Despite a computational method for the estimation of the gene-dependency level while taking account of the copy-number effect, it is experimentally demanding to determine copy numbers for cell types of interest, while it is still difficult to resolve mismatch-tolerance effect 11,16 . What's more, it has recently been reported that Cas9-induced DSBs posed obstacles to high-throughput screens in human non-transformed cells via p53-dependent cell growth arrest [17][18][19][20] . High-efficiency Cas9 editing could cause cell death in human pluripotent stem cells (hPSCs) 20 and G1 cell cycle arrest in hTERT RPE1 cells 18 .…”

Section: Introductionmentioning

confidence: 99%

Genome-wide interrogation of gene functions through base editor screens empowered by barcoded sgRNAs

Xu

¹

,

Liu

²

,

Liu

³

et al. 2020

Preprint

View full text Add to dashboard Cite

Canonical CRISPR screens rely on Cas9-induced DNA double-strand breaks (DSBs) to generate targeted gene knockouts. These DSB-dependent methodologies may yield false-positive results by mistakenly assuming targeted loci as essential for cell viability, especially when high-copy-number sites are targeted. Here, we use CRISPR cytosine base editors for genome-scale knockout screens by perturbing gene start codons or splice sites, or by introducing premature termination codons (PTCs). Combining with iBAR strategy we have previously established, we realized an iBARed cytosine Base Editing-mediated gene KnockOut (BARBEKO) screening strategy at a genome-scale (targeting 17,501 genes) in multiple human cell lines. By constructing such a cell library through lentiviral infection at a high MOI (up to 10), we significantly reduced starting cells while producing screening results with improved efficiency and accuracy. More importantly, in comparison with Cas9-mediated cell fitness screens, BARBEKO screens are no longer affected by DNA-cleavage induced cytotoxicity in HeLa, K562, or DSB-sensitive RPE1 cells. We anticipate that BARBEKO offers a valuable tool to complement the current CRISPR screens in various settings.

show abstract

“…The later study showed that Cas9 toxicity created an obstacle to the high‐throughput use of CRISPR/Cas9 for genome engineering and screening in pluripotent stem cells in response to the Cas9‐induced break. However, Bowden and coworkers 278 carried out parallel CRISPR/Cas9 screens in wild‐type and knockout hRPECs using a focused dual guide RNA library targeting 852 DNA damage response‐associated genes. They showed that although functional p53 status negatively affected identification of significantly depleted genes, optimal screen design enabled robust screen performance.…”

Section: Problems and Perspectivesmentioning

confidence: 99%

Human induced pluripotent stem cells and CRISPR/Cas-mediated targeted genome editing: Platforms to tackle sensorineural hearing loss

Stojković

¹

,

Han

²

,

Jeong

³

et al. 2021

View full text Add to dashboard Cite

Hearing loss (HL) is a major global health problem of pandemic proportions. The most common type of HL is sensorineural hearing loss (SNHL) which typically occurs when cells within the inner ear are damaged. Human induced pluripotent stem cells (hiPSCs) can be generated from any individual including those who suffer from different types of HL. The development of new differentiation protocols to obtain cells of the inner ear including hair cells (HCs) and spiral ganglion neurons (SGNs) promises to expedite cell-based therapy and screening of potential pharmacologic and genetic therapies using human models. Considering age-related, acoustic, ototoxic, and genetic insults which are the most frequent causes of irreversible damage of HCs and SGNs, new methods of genome editing (GE), especially the CRISPR/Cas9 technology, could bring additional opportunities to understand the pathogenesis of human SNHL and identify novel therapies. However, important challenges associated with both hiPSCs and GE need to be overcome before scientific discoveries are correctly translated to effective and patient-safe applications. The purpose of the present review is (a) to summarize the findings from published reports utilizing hiPSCs for studies of SNHL, hence complementing recent reviews focused on animal studies, and (b) to outline promising future directions for deciphering SNHL using disruptive molecular and genomic technologies.

show abstract

Parallel CRISPR-Cas9 screens clarify impacts of p53 on screen performance

Cited by 12 publications

References 29 publications

Genome-wide interrogation of gene functions through base editor screens empowered by barcoded sgRNAs

Genome-wide interrogation of gene functions through base editor screens empowered by barcoded sgRNAs

Genome-wide interrogation of gene functions through base editor screens empowered by barcoded sgRNAs

Human induced pluripotent stem cells and CRISPR/Cas-mediated targeted genome editing: Platforms to tackle sensorineural hearing loss

Contact Info

Product

Resources

About