Engineering and study of protein function by directed evolution has been limited by the requirement to introduce DNA libraries of defined size or to use global mutagenesis. Here, we develop a strategy to repurpose the somatic hypermutation machinery used in antibody affinity maturation to efficiently perform protein engineering in situ. Using catalytically inactive Cas9 (dCas9) to recruit variants of the deaminase AID (CRISPR-X), we can specifically mutagenize endogenous targets with limited off-target damage. This generates diverse libraries of localized point mutations, in contrast to insertions and deletions created by active Cas9, and can be used to mutagenize multiple genomic locations simultaneously. With this technology, we mutagenize GFP and select for spectrum-shifted variants, including EGFP. In addition, we mutate the target of the cancer therapeutic bortezomib, PSMB5, and identify known and novel mutations that confer resistance to treatment. Finally, we utilize a hyperactive AID variant with dramatically increased activity to mutagenize endogenous loci both upstream and downstream of transcriptional start sites. These experiments illustrate a powerful new approach to create highly complex libraries of genetic variants in native context, which can be broadly applied to investigate and improve protein function.

CRISPR-Cas9 screens are powerful tools for high-throughput interrogation of genome function, but can be confounded by nuclease-induced toxicity at both on-and off-target sites, likely due to DNA damage. Here, to test potential solutions to this issue, we design and analyse a CRISPR-Cas9 library with 10 variable-length guides per gene and thousands of negative controls targeting non-functional, non-genic regions (termed safe-targeting guides), in addition to non-targeting controls. We find this library has excellent performance in identifying genes affecting growth and sensitivity to the ricin toxin. The safe-targeting guides allow for proper control of toxicity from on-target DNA damage. Using this toxicity as a proxy to measure off-target cutting, we demonstrate with tens of thousands of guides both the nucleotide position-dependent sensitivity to single mismatches and the reduction of off-target cutting using truncated guides. Our results demonstrate a simple strategy for high-throughput evaluation of target specificity and nuclease toxicity in Cas9 screens.

Identification of effective combination therapies is critical to address the emergence of drug-resistant cancers, but direct screening of all possible drug combinations is infeasible. Here we introduce a CRISPR-based double knockout (CDKO) system that improves the efficiency of combinatorial genetic screening using an effective strategy for cloning and sequencing paired single-guide RNA libraries and a robust statistical scoring method for calculating genetic interactions (GIs) from CRISPR-deleted gene pairs. We applied CDKO to generate a large-scale human GI map, comprising 490,000 double-sgRNAs directed against 21,321 pairs of drug targets in K562 leukemia cells and identified synthetic lethal drug target pairs for which corresponding drugs exhibit synergistic killing. These included the BCL2L1 and MCL1 combination, which was also effective in imatinib-resistant cells. We further validated this system by identifying known and previously unidentified GIs between modifiers of ricin toxicity. This work provides an effective strategy to screen synergistic drug combinations at high-throughput and a CRISPR-based tool to dissect functional GI networks.

antagonist ͉ cancer ͉ Gli ͉ medulloblastoma

Cancer genomics studies have nominated thousands of putative cancer driver genes 1 ; a major challenge is to develop high-throughput and accurate models to define their functions. Here we devised a scalable cancer spheroid model and performed genome-wide CRISPR screens in 2D-monolayers and 3D lung cancer spheroids. CRISPR phenotypes in 3D more accurately recapitulate those of in vivo tumors, and genes with differential sensitivities between 2D and 3D are strongly enriched for significant mutations in lung cancers. These analyses also revealed novel drivers essential for cancer growth in 3D and in vivo , but not in 2D. Notably, we discovered that CPD (Carboxypeptidase D) is responsible for removal of a c-terminal RKRR motif 2 of IGF1R α-chain, critical for receptor activity. CPD expression correlates with patient outcomes in lung cancer, and loss of CPD reduced tumor growth. Our results reveal key differences between 2D and 3D cancer models, and establish a generalizable strategy to perform CRISPR screens in spheroids to uncover cancer vulnerabilities.

No abstract

The primary cilium organizes Hedgehog signaling and shapes embryonic development, and its dysregulation is the unifying cause of ciliopathies. We conducted a functional genomic screen for Hedgehog signaling by engineering antibiotic-based selection of Hedgehog-responsive cells and applying genome-wide CRISPR-mediated gene disruption. The screen robustly identifies factors required for ciliary signaling with few false positives or false negatives. Characterization of hit genes uncovers novel components of several ciliary structures, including a protein complex containing δ- and ε-tubulin that is required for centriole maintenance. The screen also provides an unbiased tool for classifying ciliopathies and reveals that many congenital heart disorders are caused by loss of ciliary signaling. Collectively, our study enables a systematic analysis of ciliary function and of ciliopathies and also defines a versatile platform for dissecting signaling pathways through CRISPR-based screening.

No abstract