Ph.D., Harvard University, Biophysics (2012)
S.M., Harvard University, Applied Mathematics (2008)
A.B., Harvard University, Chemistry and Physics (2006)
Michael Bassik, Postdoctoral Faculty Sponsor
Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells.
Engineering and study of protein function by directed evolution has been limited by the technical requirement to use global mutagenesis or introduce DNA libraries. Here, we develop CRISPR-X, a strategy to repurpose the somatic hypermutation machinery for protein engineering in situ. Using catalytically inactive dCas9 to recruit variants of cytidine deaminase (AID) with MS2-modified sgRNAs, we can specifically mutagenize endogenous targets with limited off-target damage. This generates diverse libraries of localized point mutations and can target multiple genomic locations simultaneously. We mutagenize GFP and select for spectrum-shifted variants, including EGFP. Additionally, we mutate the target of the cancer therapeutic bortezomib, PSMB5, and identify known and novel mutations that confer bortezomib resistance. Finally, using a hyperactive AID variant, we mutagenize loci both upstream and downstream of transcriptional start sites. These experiments illustrate a powerful approach to create complex libraries of genetic variants in native context, which is broadly applicable to investigate and improve protein function.
View details for DOI 10.1038/nmeth.4038
View details for PubMedID 27798611
M13 Bacteriophage Display Framework That Allows Sortase-Mediated Modification of Surface-Accessible Phage Proteins
2012; 23 (7): 1478-1487
We exploit bacterial sortases to attach a variety of moieties to the capsid proteins of M13 bacteriophage. We show that pIII, pIX, and pVIII can be functionalized with entities ranging from small molecules (e.g., fluorophores, biotin) to correctly folded proteins (e.g., GFP, antibodies, streptavidin) in a site-specific manner, and with yields that surpass those of any reported using phage display technology. A case in point is modification of pVIII. While a phage vector limits the size of the insert into pVIII to a few amino acids, a phagemid system limits the number of copies actually displayed at the surface of M13. Using sortase-based reactions, a 100-fold increase in the efficiency of display of GFP onto pVIII is achieved. Taking advantage of orthogonal sortases, we can simultaneously target two distinct capsid proteins in the same phage particle and maintain excellent specificity of labeling. As demonstrated in this work, this is a simple and effective method for creating a variety of structures, thus expanding the use of M13 for materials science applications and as a biological tool.
View details for DOI 10.1021/bc300130z
View details for Web of Science ID 000306452500015
View details for PubMedID 22759232
Genome-scale measurement of off-target activity using Cas9 toxicity in high-throughput screens
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.
View details for DOI 10.1038/ncomms15178
View details for Web of Science ID 000400851300001
View details for PubMedID 28474669
Synergistic drug combinations for cancer identified in a CRISPR screen for pairwise genetic interactions
2017; 35 (5): 463-?
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 (sgRNA) 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 in high-throughput and a CRISPR-based tool to dissect functional GI networks.
View details for DOI 10.1038/nbt.3834
View details for Web of Science ID 000400809800021
View details for PubMedID 28319085
Population- and individual- specific regulatory variation in Sardinia
2017; 49 (5): 700-?
Genetic studies of complex traits have mainly identified associations with noncoding variants. To further determine the contribution of regulatory variation, we combined whole-genome and transcriptome data for 624 individuals from Sardinia to identify common and rare variants that influence gene expression and splicing. We identified 21,183 expression quantitative trait loci (eQTLs) and 6,768 splicing quantitative trait loci (sQTLs), including 619 new QTLs. We identified high-frequency QTLs and found evidence of selection near genes involved in malarial resistance and increased multiple sclerosis risk, reflecting the epidemiological history of Sardinia. Using family relationships, we identified 809 segregating expression outliers (median z score of 2.97), averaging 13.3 genes per individual. Outlier genes were enriched for proximal rare variants, providing a new approach to study large-effect regulatory variants and their relevance to traits. Our results provide insight into the effects of regulatory variants and their relationship to population history and individual genetic risk.
View details for DOI 10.1038/ng.3840
View details for Web of Science ID 000400051400010
View details for PubMedID 28394350
Translation readthrough mitigation
2016; 534 (7609): 719-?
A fraction of ribosomes engaged in translation will fail to terminate when reaching a stop codon, yielding nascent proteins inappropriately extended on their C termini. Although such extended proteins can interfere with normal cellular processes, known mechanisms of translational surveillance are insufficient to protect cells from potential dominant consequences. Here, through a combination of transgenics and CRISPR–Cas9 gene editing in Caenorhabditis elegans, we demonstrate a consistent ability of cells to block accumulation of C-terminal-extended proteins that result from failure to terminate at stop codons. Sequences encoded by the 3′ untranslated region (UTR) were sufficient to lower protein levels. Measurements of mRNA levels and translation suggested a co- or post-translational mechanism of action for these sequences in C. elegans. Similar mechanisms evidently operate in human cells, in which we observed a comparable tendency for translated human 3′ UTR sequences to reduce mature protein expression in tissue culture assays, including 3′ UTR sequences from the hypomorphic ‘Constant Spring’ haemoglobin stop codon variant. We suggest that 3′ UTRs may encode peptide sequences that destabilize the attached protein, providing mitigation of unwelcome and varied translation errors.
View details for DOI 10.1038/nature18308
View details for Web of Science ID 000378676000044
View details for PubMedID 27281202
View details for PubMedCentralID PMC5054982
Orthogonal Labeling of M13 Minor Capsid Proteins with DNA to Self-Assemble End-to-End Multiphage Structures
ACS SYNTHETIC BIOLOGY
2013; 2 (9): 490-496
M13 bacteriophage has been used as a scaffold to organize materials for various applications. Building more complex multiphage devices requires precise control of interactions between the M13 capsid proteins. Toward this end, we engineered a loop structure onto the pIII capsid protein of M13 bacteriophage to enable sortase-mediated labeling reactions for C-terminal display. Combining this with N-terminal sortase-mediated labeling, we thus created a phage scaffold that can be labeled orthogonally on three capsid proteins: the body and both ends. We show that covalent attachment of different DNA oligonucleotides at the ends of the new phage structure enables formation of multiphage particles oriented in a specific order. These have potential as nanoscale scaffolds for multi-material devices.
View details for DOI 10.1021/sb4000195
View details for Web of Science ID 000324842100002
View details for PubMedID 23713956
Cellular binding, motion, and internalization of synthetic gene delivery polymers
BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH
2007; 1773 (10): 1583-1588
Using fluorescence microscopy we have tracked the cellular binding, surface motion, and internalization of polyarginine and polyethylenimine, cationic ligands used for gene and protein delivery. Each ligand was complexed with a quantum dot to provide a photostable probe. Transfection with exogenous DNA was used to relate the observed motion to gene delivery. Cell surface motion was independent of sulfated proteoglycans, but dependent on cholesterol. Cellular internalization required sulfated proteoglycans and cholesterol. These observations suggest that sulfated proteoglycans act as cellular receptors for the cationic ligands, rather than only passive binding sites. Understanding the interaction of polyarginine and polyethylenimine with the plasma membrane may assist in designing more efficient gene delivery systems.
View details for DOI 10.1016/j.bbamcr.2007.07.009
View details for Web of Science ID 000250528400009
View details for PubMedID 17888530