Base editing screens map mutations affecting interferon-γ signaling in cancer

Coelho, Matthew A.; Cooper, Sarah; Strauß, Magdalena E; Karakoç, Emre; Bhosle, Shriram G.; Gonçalvès, Emanuel; Picco, Gabriele; Burgold, Thomas; Cattaneo, Chiara; Veninga, Vivien; Consonni, Sarah; Dinçer, Cansu; Vieira, Sara F; Gibson, Freddy; Barthorpe, Syd; Hardy, Claire; Rein, Joel; Thomas, Mark; Marioni, John C.; Voest, Emile E.; Bassett, Andrew; Garnett, Mathew J.

doi:10.1016/j.ccell.2022.12.009

Cited by 42 publications

(42 citation statements)

References 87 publications

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“…For granular accounting of variants that enabled growth in the presence of erlotinib or osimertinib after editing with AID-dCas9 we performed a secondary screen using select sgRNAs identified in the primary screen coupled to a single cell genotyping readout (Figure 4A). Single cell (sc) genotyping was performed using the Tapestri platform, which has been applied previously to interrogate genome editing outcomes in a screening context (29, 69, 70). Vectors expressing the most significantly enriched sgRNAs for EGFR regions 1-4 and BRAF regions 1-2 were pooled, along with the weak hit sgRNAs.…”

Section: Resultsmentioning

confidence: 99%

Accelerated drug resistant variant discovery with an enhanced, scalable mutagenic base editor platform

Dorighi,

Zhu,

Fortin

et al. 2023

Preprint

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Personalized cancer therapeutics bring directed treatment options to patients based on the genetic signatures of their tumors. Unfortunately, tumor genomes are remarkably adaptable, and acquired resistance to these drugs through genetic means is an all-too-frequent occurrence. Identifying mutations that promote resistance within drug-treated patient populations can be cost, resource, and time intensive. Accordingly, base editing, enabled by Cas9-deaminase domain fusions, has emerged as a promising approach for rapid, large-scale resistance variant screening in situ. We adapted and optimized a conditional activation-induced cytidine deaminase (AID)-dCas9 system, which demonstrated greater heterogeneity of edits with an expanded footprint compared to the most commonly utilized cytosine base editor, BE4. When combined with a custom sgRNA library, we were able to identify both individual and complex, compound variants in EGFR and BRAF that confer resistance to established EGFR inhibitors. This system and the developed analytical pipeline provide a simple, highly-scalable platform for cis or trans drug-modifying variant discovery and for uncovering unique insights into protein structure-function relationships.

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Section: Resultsmentioning

confidence: 99%

Accelerated drug resistant variant discovery with an enhanced, scalable mutagenic base editor platform

Dorighi,

Zhu,

Fortin

et al. 2023

Preprint

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“…Although CRISPRa has been widely used for GOF mutations in preclinical studies, 17,30 the requirement of long‐term presence of CRISPR components raises safety concerns, making it less ideal for clinical practices. However, base editing 136 and prime editing 13 hold significant potential for introducing GOF mutations without causing DSBs, offering a safer approach to generating therapeutic T‐cell products with enhanced functionality. To fully harness the potential of GOF mutations, an important direction of future work should aim at identifying targets that can significantly boost T‐cell functionality.…”

Section: Conclusion and Future Directionmentioning

confidence: 99%

“…Although CRISPRa has been widely used for GOF mutations in preclinical studies, 17,30 the requirement of long-term presence of CRISPR components raises safety concerns, making it less ideal for clinical practices. However, base editing 136 and prime editing 13 One of the significant obstacles to achieving effective antitumor efficacy of genome-edited T cells is their poor persistence in vivo. Although several factors, such as exhaustion and hostile tumor microenvironment, could contribute to this issue, it is essential to consider the activation of endogenous immunity against CRISPR-Cas peptides presented by the edited T cells, given that CRISPR-Cas systems are derived from pathogenic bacteria.…”

Section: New Screening Strategies To Address Old Questionsmentioning

confidence: 99%

Applications of CRISPR technology in cellular immunotherapy

Zhou

Renauer

Zhou

et al. 2023

Immunological Reviews

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SummaryCRISPR technology has transformed multiple fields, including cancer and immunology. CRISPR‐based gene editing and screening empowers direct genomic manipulation of immune cells, opening doors to unbiased functional genetic screens. These screens aid in the discovery of novel factors that regulate and reprogram immune responses, offering novel drug targets. The engineering of immune cells using CRISPR has sparked a transformation in the cellular immunotherapy field, resulting in a multitude of ongoing clinical trials. In this review, we discuss the development and applications of CRISPR and related gene editing technologies in immune cells, focusing on functional genomics screening, gene editing‐based cell therapies, as well as future directions in this rapidly advancing field.

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“…12 Base editing is a form of precision genome editing that has enabled the direct mutational scanning of a variety of endogenous proteins. [13][14][15][16] Base editing has the advantage of targeting the native gene, avoiding the confounding effects of overexpressing mutants of a protein of interest. By coupling a fluorescence-based DNA methylation reporter system with base editing to generate a library of mutants, we previously identified a wide variety of LOF mutations in DNMT3A, and highlighted the importance of the PWWP domain for enzyme activity, particularly in its ability to bind to DNA.…”

Section: Introductionmentioning

confidence: 99%

Base Editor Scanning Reveals Activating Mutations of DNMT3A

García

Lue

Liang

et al. 2023

Preprint

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DNA methyltransferase 3A (DNMT3A) is ade novocytosine methyltransferase responsible for establishing proper DNA methylation during mammalian development. Loss-of-function (LOF) mutations to DNMT3A, including the hotspot mutation R882H, frequently occur in developmental growth disorders and hematological diseases, including clonal hematopoiesis (CH) and acute myeloid leukemia (AML). Accordingly, identifying mechanisms that activate DNMT3A is of both fundamental and therapeutic interest. Here, we applied a base editor mutational scanning strategy with an improved DNA methylation reporter to systematically identify DNMT3A activating mutations in cells. By integrating an optimized cellular recruitment strategy with paired isogenic cell lines with or without the LOF hotspot R882H mutation, we identify and validate three distinct hyperactivating mutations within or interacting with the regulatory ADD domain of DNMT3A, nominating these regions as potential functional target sites for pharmacological intervention. Notably, these mutations are still activating in the context of a heterozygous R882H mutation. Altogether, we showcase the utility of base editor scanning for discovering functional regions of target proteins.SynopsisUsing base editor mutagenesis and a DNA methylation reporter optimized to find activating mutations, we identify novel hyperactivating mutations in DNMT3A that suggest new mechanisms of allosteric control.

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Base editing screens map mutations affecting interferon-γ signaling in cancer

Cited by 42 publications

References 87 publications

Accelerated drug resistant variant discovery with an enhanced, scalable mutagenic base editor platform

Accelerated drug resistant variant discovery with an enhanced, scalable mutagenic base editor platform

Applications of CRISPR technology in cellular immunotherapy

Base Editor Scanning Reveals Activating Mutations of DNMT3A

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