CRISPR library designer (CLD): software for multispecies design of single guide RNA libraries

Table S9. Software used in this study. (XLSX 39 kb

Data-analysis strategies for image-based cell profiling

Image-based cell profiling is a high-throughput strategy for the quantification of phenotypic differences among a variety of cell populations. It paves the way to studying biological systems on a large scale by using chemical and genetic perturbations. The general workflow for this technology involves image acquisition with high-throughput microscopy systems and subsequent image processing and analysis. Here, we introduce the steps required to create high-quality image-based (i.e., morphological) profiles from a collection of microscopy images. We recommend techniques that have proven useful in each stage of the data analysis process, on the basis of the experience of 20 laboratories worldwide that are refining their image-based cell-profiling methodologies in pursuit of biological discovery. The recommended techniques cover alternatives that may suit various biological goals, experimental designs, and laboratories' preferences

Characterization of the mammalian family of DCN-type NEDD8 E3 ligases

Cullin-RING ligases (CRL) are ubiquitin E3s that bind substrates through variable substrate-receptor proteins. CRLs are activated by attachment of the ubiquitin-like protein NEDD8 to the Cullin subunit and DCNs are NEDD8 E3 ligases that promote neddylation. Mammalian cells express five DCN-like proteins and little is known about their specific functions or interaction partners. We found that DCNLs form stable stoichiometric complexes with CAND1 and Cullins that can only be neddylated in the presence of substrate adaptor. These DCNL-CUL-CAND1 complexes may represent “reserve” CRLs that can be rapidly activated when needed. We further found that all DCNLs interact with most Cullin subtypes, but that they are likely responsible for the neddylation of different subpopulations of any given Cullin. This is consistent with the fact that the subcellular localization of DCNLs in tissue culture cells differs and that they show unique tissue specific expression patterns in mice. Thus, the specificity between DCNL-type NEDD8 E3 enzymes and their Cullin substrates is only apparent in well-defined physiological contexts and related to their subcellular distribution and restricted expression

Noncanonical function of DGCR8 controls mESC exit from pluripotency

Mouse embryonic stem cells (mESCs) deficient for DGCR8, a key component of the microprocessor complex, present strong differentiation defects. However, the exact reasons impairing their commitment remain elusive. The analysis of newly generated mutant mESCs revealed that DGCR8 is essential for the exit from the pluripotency state. To dissociate canonical versus noncanonical functions of DGCR8, we complemented the mutant mESCs with a phosphomutant DGCR8, which restored microRNA levels but did not rescue the exit from pluripotency defect. Integration of omics data and RNA immunoprecipitation experiments established DGCR8 as a direct interactor of Tcf7l1 mRNA, a core component of the pluripotency network. Finally, we found that DGCR8 facilitated the splicing of Tcf7l1, an event necessary for the differentiation of mESCs. Our data reveal a new noncanonical function of DGCR8 in the modulation of the alternative splicing of Tcf7l1 mRNA in addition to its established function in microRNA biogenesis

CRISPR-Cas9 ribonucleoprotein-mediated co-editing and counterselection in the rice blast fungus

The rice blast fungus Magnaporthe oryzae is the most serious pathogen of cultivated rice and a significant threat to global food security. To accelerate targeted mutation and specific genome editing in this species, we have developed a rapid plasmid-free CRISPR-Cas9-based genome editing method. We show that stable expression of Cas9 is highly toxic to M. oryzae. However efficient gene editing can be achieved by transient introduction of purified Cas9 pre-complexed to RNA guides to form ribonucleoproteins (RNPs). When used in combination with oligonucleotide or PCR-generated donor DNAs, generation of strains with specific base pair edits, in-locus gene replacements, or multiple gene edits, is very rapid and straightforward. We demonstrate a co-editing strategy for the creation of single nucleotide changes at specific loci. Additionally, we report a novel counterselection strategy which allows creation of precisely edited fungal strains that contain no foreign DNA and are completely isogenic to the wild type. Together, these developments represent a scalable improvement in the precision and speed of genetic manipulation in M. oryzae and are likely to be broadly applicable to other fungal species

Modified penetrance of coding variants by cis-regulatory variation contributes to disease risk

Coding variants represent many of the strongest associations between genotype and phenotype; however, they exhibit interindividual differences in effect, termed 'variable penetrance'. Here, we study how cis-regulatory variation modifies the penetrance of coding variants. Using functional genomic and genetic data from the Genotype-Tissue Expression Project (GTEx), we observed that in the general population, purifying selection has depleted haplotype combinations predicted to increase pathogenic coding variant penetrance. Conversely, in cancer and autism patients, we observed an enrichment of penetrance increasing haplotype configurations for pathogenic variants in disease-implicated genes, providing evidence that regulatory haplotype configuration of coding variants affects disease risk. Finally, we experimentally validated this model by editing a Mendelian single-nucleotide polymorphism (SNP) using CRISPR/Cas9 on distinct expression haplotypes with the transcriptome as a phenotypic readout. Our results demonstrate that joint regulatory and coding variant effects are an important part of the genetic architecture of human traits and contribute to modified penetrance of disease-causing variants.Peer reviewe

Data-analysis strategies for image-based cell profiling

Image-based cell profiling is a high-throughput strategy for the quantification of phenotypic differences among a variety of cell populations. It paves the way to studying biological systems on a large scale by using chemical and genetic perturbations. The general workflow for this technology involves image acquisition with high-throughput microscopy systems and subsequent image processing and analysis. Here, we introduce the steps required to create high-quality image-based (i.e., morphological) profiles from a collection of microscopy images. We recommend techniques that have proven useful in each stage of the data analysis process, on the basis of the experience of 20 laboratories worldwide that are refining their image-based cell-profiling methodologies in pursuit of biological discovery. The recommended techniques cover alternatives that may suit various biological goals, experimental designs, and laboratories' preferences.Peer reviewe