CRISPR/Cas9 screening using unique molecular identifiers

Schmierer, Bernhard; Botla, Sandeep K.; Zhang, Jilin; Turunen, Mikko; Kivioja, Teemu; Taipale, Jussi

doi:10.15252/msb.20177834

Cited by 63 publications

(72 citation statements)

References 23 publications

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“…Rather than sequencing guides directly, we used arbitrary nucleotide barcodes embedded in the guide RNA expression plasmid. One major advantage of sequencing these barcodes is that each guide can be linked to a few different barcodes, providing replicate measurements of its effect within a single experiment (Michlits et al, 2017;Schmierer et al, 2017). In contrast, direct guide sequencing cannot distinguish between independently transformed lineages within a single experiment.…”

Section: Linear Amplification Of Guide-linked Nucleotide Barcodes By mentioning

confidence: 99%

A genome-scale CRISPR interference guide library enables comprehensive phenotypic profiling in yeast

McGlincy

Meacham

Swain

et al. 2020

Preprint

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CRISPR/Cas9-mediated transcriptional interference (CRISPRi) enables programmable gene knock-down, yielding interpretable loss-of-function phenotypes for nearly any gene. Effective, inducible CRISPRi has been demonstrated in budding yeast, but no genome-scale guide libraries have been reported. We present a comprehensive yeast CRISPRi library, based on empirical design rules, containing 10 distinct guides for most genes. Competitive growth after pooled transformation revealed strong fitness defects for most essential genes, verifying that the library provides comprehensive genome coverage. We used the relative growth defects caused by different guides targeting essential genes to further refine yeast CRISPRi design rules. In order to obtain more accurate and robust guide abundance measurements in pooled screens, we link guides with random nucleotide barcodes and carry out linear amplification by in vitro transcription. Taken together, we demonstrate a broadly useful platform for comprehensive, high-precision CRISPRi screening in yeast.

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Section: Linear Amplification Of Guide-linked Nucleotide Barcodes By mentioning

confidence: 99%

A genome-scale CRISPR interference guide library enables comprehensive phenotypic profiling in yeast

McGlincy

Meacham

Swain

et al. 2020

Preprint

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“…In MPRA, for example, promoter or enhancer variants are typically tagged with a transcribed barcode, which is then used to infer the identity of the variant that led to expression changes 11 . Similarly, screening approaches that use unique molecular identifiers (UMIs) to obtain an absolute count of cells receiving a perturbation such as an sgRNA may be susceptible to uncoupling between the UMI and the sgRNA, potentially leading to an inflated estimate of diversity 12,13 . Recently, numerous approaches to combinatorial CRISPR screens have been described, for which accurate quantitation of two unique sgRNA sequences in the same vector presents the same challenge [14][15][16][17][18] .…”

Section: Introductionmentioning

confidence: 99%

Uncoupling of sgRNAs from their associated barcodes during PCR amplification of combinatorial CRISPR screens

Hegde

Strand

Hanna

et al. 2018

Preprint

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Many implementations of pooled screens in mammalian cells rely on linking an element of interest to a barcode, with the latter subsequently quantitated by next generation sequencing. However, substantial uncoupling between these paired elements during lentiviral production has been reported, especially as the distance between elements increases. We detail that PCR amplification is another major source of uncoupling, and becomes more pronounced with increased amounts of DNA template molecules and PCR cycles. To lessen uncoupling in systems that use paired elements for detection, we recommend minimizing the distance between elements, using low and equal template DNA inputs for plasmid and genomic DNA during PCR, and minimizing the number of PCR cycles. We also present a vector design for conducting combinatorial CRISPR screens that enables accurate barcode-based detection with a single short sequencing read and minimal uncoupling.

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“…1a,b). Shuffle-Seq uses Unique Transduction Barcodes (UTBs), similar to those used in previous CRISPR screens 20,21 , such that the probability of two cells getting the same combination of perturbation-UTBs is extremely low. Multiple perturbations are delivered into the cell through one or more rounds of viral transduction.…”

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confidence: 99%

Shuffle-Seq: En masse combinatorial encoding for n-way genetic interaction screens

Dixit

Kuksenko

Feldman

et al. 2019

Preprint

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Genetic interactions, defined as the non-additive phenotypic impact of combinations of genes, are a hallmark of the mapping from genotype to phenotype. However, genetic interactions remain challenging to systematically test given the massive number of possible combinations. In particular, while large-scale screening efforts in yeast have quantified pairwise interactions that affect cell viability, or synthetic lethality, between all pairs of genes as well as for a limited number of three-way interactions, it has previously been intractable to perform the large screens needed to comprehensively assess interactions in a mammalian genome. Here, we develop Shuffle-Seq, a scalable method to assay genetic interactions. Shuffle-Seq leverages the co-inheritance of genetically encoded barcodes in dividing cells and can scale in proportion to sequencing throughput. We demonstrate the technical validity of Shuffle-Seq and apply it to screening for mechanisms underlying drug resistance in a melanoma model. Shuffle-Seq should allow screens of hundreds of millions of combinatorial perturbations and facilitate the understanding of genetic dependencies and drug sensitivities.

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CRISPR/Cas9 screening using unique molecular identifiers

Cited by 63 publications

References 23 publications

A genome-scale CRISPR interference guide library enables comprehensive phenotypic profiling in yeast

A genome-scale CRISPR interference guide library enables comprehensive phenotypic profiling in yeast

Uncoupling of sgRNAs from their associated barcodes during PCR amplification of combinatorial CRISPR screens

Shuffle-Seq: En masse combinatorial encoding for n-way genetic interaction screens

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