Modular vector assembly enables rapid assessment of emerging CRISPR technologies.

The diversity of CRISPR systems, coupled with scientific ingenuity, has led to an explosion of applications; however, to test newly described innovations in their model systems, researchers typically embark on cumbersome, one-off cloning projects to generate custom reagents that are optimized for their biological questions. Here, we leverage Golden Gate cloning to create the Fragmid toolkit, a modular set of CRISPR cassettes and delivery technologies, along with a web portal, resulting in a combinatorial platform that enables scalable vector assembly within days. We further demonstrate that multiple CRISPR technologies can be assessed in parallel in a pooled screening format using this resource, enabling the rapid optimization of both novel technologies and cellular models. These results establish Fragmid as a robust system for the rapid design of CRISPR vectors, and we anticipate that this assembly approach will be broadly useful for systematic development, comparison, and dissemination of CRISPR technologies.

Modular vector assembly enables rapid assessment of emerging CRISPR technologies.

The diversity of CRISPR systems, coupled with scientific ingenuity, has led to an explosion of applications; however, to test newly-described innovations in their model systems, researchers typically embark on cumbersome, one-off cloning projects to generate custom reagents that are optimized for their biological questions. Here, we leverage Golden Gate cloning to create the Fragmid toolkit, a modular set of CRISPR cassettes and delivery technologies, along with a web portal, resulting in a combinatorial platform that enables scalable vector assembly within days. We further demonstrate that multiple CRISPR technologies can be assessed in parallel in a pooled screening format using this resource, enabling the rapid optimization of both novel technologies and cellular models. These results establish Fragmid as a robust system for the rapid design of CRISPR vectors, and we anticipate that this assembly approach will be broadly useful for systematic development, comparison, and dissemination of CRISPR technologies.

Optimization of Cas12a for multiplexed genome-scale transcriptional activation.

Cas12a CRISPR technology, unlike Cas9, allows for facile multiplexing of guide RNAs from a single transcript, simplifying combinatorial perturbations. While Cas12a has been implemented for multiplexed knockout genetic screens, it has yet to be optimized for CRISPR activation (CRISPRa) screens in human cells. Here, we develop a new Cas12a-based transactivation domain (TAD) recruitment system using the ALFA nanobody and demonstrate simultaneous activation of up to four genes. We screen a genome-wide library to identify modulators of growth and MEK inhibition, and we compare these results with those obtained with open reading frame (ORF) overexpression and Cas9-based CRISPRa. We find that the activity of multiplexed arrays is largely predictable from the best-performing guide and provide criteria for selecting active guides. We anticipate that these results will greatly accelerate the exploration of gene function and combinatorial phenotypes at scale.

Telomerase inhibition is an effective therapeutic strategy in TERT promoter-mutant glioblastoma models with low tumor volume.

BACKGROUND: Glioblastoma is one of the most lethal forms of cancer, with 5-year survival rates of only 6%. Glioblastoma-targeted therapeutics have been challenging to develop due to significant inter- and intra-tumoral heterogeneity. Telomerase reverse transcriptase gene (TERT) promoter mutations are the most common known clonal oncogenic mutations in glioblastoma. Telomerase is therefore considered to be a promising therapeutic target against this tumor. However, an important limitation of this strategy is that cell death does not occur immediately after telomerase ablation, but rather after several cell divisions required to reach critically short telomeres. We, therefore, hypothesize that telomerase inhibition would only be effective in glioblastomas with low tumor burden. METHODS: We used CRISPR interference to knock down TERT expression in TERT promoter-mutant glioblastoma cell lines and patient-derived models. We then measured viability using serial proliferation assays. We also assessed for features of telomere crisis by measuring telomere length and chromatin bridge formation. Finally, we used a doxycycline-inducible CRISPR interference system to knock down TERT expression in vivo early and late in tumor development. RESULTS: Upon TERT inactivation, glioblastoma cells lose their proliferative ability over time and exhibit telomere shortening and chromatin bridge formation. In vivo, survival is only prolonged when TERT knockdown is induced shortly after tumor implantation, but not when the tumor burden is high. CONCLUSIONS: Our results support the idea that telomerase inhibition would be most effective at treating glioblastomas with low tumor burden, for example in the adjuvant setting after surgical debulking and chemoradiation.

Benchmarking of SpCas9 variants enables deeper base editor screens of BRCA1 and BCL2.

Numerous rationally-designed and directed-evolution variants of SpCas9 have been reported to expand the utility of CRISPR technology. Here, we assess the activity and specificity of WT-Cas9 and 10 SpCas9 variants by benchmarking their PAM preferences, on-target activity, and off-target susceptibility in cell culture assays with thousands of guides targeting endogenous genes. To enhance the coverage and thus utility of base editing screens, we demonstrate that the SpCas9-NG and SpG variants are compatible with both A > G and C > T base editors, more than tripling the number of guides and assayable residues. We demonstrate the performance of these technologies by screening for loss-of-function mutations in BRCA1 and Venetoclax-resistant mutations in BCL2, identifying both known and new mutations that alter function. We anticipate that the tools and methodologies described here will facilitate the investigation of genetic variants at a finer and deeper resolution for any locus of interest.

Massively parallel assessment of human variants with base editor screens.

Understanding the functional consequences of single-nucleotide variants is critical to uncovering the genetic underpinnings of diseases, but technologies to characterize variants are limiting. Here, we leverage CRISPR-Cas9 cytosine base editors in pooled screens to scalably assay variants at endogenous loci in mammalian cells. We benchmark the performance of base editors in positive and negative selection screens, identifying known loss-of-function mutations in BRCA1 and BRCA2 with high precision. To demonstrate the utility of base editor screens to probe small molecule-protein interactions, we screen against BH3 mimetics and PARP inhibitors, identifying point mutations that confer drug sensitivity or resistance. We also create a library of single guide RNAs (sgRNAs) predicted to generate 52,034 ClinVar variants in 3,584 genes and conduct screens in the presence of cellular stressors, identifying loss-of-function variants in numerous DNA damage repair genes. We anticipate that this screening approach will be broadly useful to readily and scalably functionalize genetic variants.