Rational design and whole-genome predictions of single guide RNAs for efficient CRISPR/Cas9-mediated genome editing in Ciona

The CRISPR/Cas9 system has emerged as an important tool for a wide variety of genome engineering applications, including reverse genetic screens. Previously, we described the implementation of the CRISPR/Cas9 system to induce tissue-specific mutations at targeted locations in the genome of the sea squirt Ciona (STOLFI et al. 2014). In the present study, we designed 83 single guide RNA (sgRNA) vectors targeting 23 genes expressed in the cardiopharyngeal progenitors and surrounding tissues in the Ciona embryo and measured their mutagenesis efficacy rates by massively parallel indel detection at the targeted loci using highthroughput sequencing. We show that the combined activity of two highly active sgRNAs allows us to generate large (>3 kbp) deletions of intervening genomic DNA in somatic cells of electroporated embryos, permitting tissue-specific gene knockouts. Additionally, we employed L1-regularized regression modeling to develop an optimal sgRNA design algorithm (TuniCUT), based on correlations between target sequence features and mutagenesis rates. Using this algorithm, we have predicted mutagenesis rates for sgRNAs targeting all 4,853,589 sites in the Ciona genome, which we have compiled into a "CRISPR/Cas9-induced Ciona Knock-Out" (Ci2KO) sgRNA sequence library. Finally, we describe a new method for the assembly of sgRNA expression cassettes using a simple one-step overlap PCR (OSO-PCR) protocol. These cassettes can be electroporated directly into Ciona embryos as unpurified PCR products to drive sgRNA expression, bypassing the need for time-consuming cloning and plasmid DNA preparations. We anticipate that this method will be used in combination with genome-wide sgRNA predictions to systematically investigate tissue-specific gene functions in Ciona

Coincident generation of pyramidal neurons and protoplasmic astrocytes in neocortical columns

Astrocytes, one of the most common cell types in the brain, are essential for processes ranging from neural development through potassium homeostasis to synaptic plasticity. Surprisingly, the developmental origins of astrocytes in the neocortex are still controversial. To investigate the patterns of astrocyte development in the neocortex we examined cortical development in a transgenic mouse in which a random, sparse subset of neural progenitors undergoes CRE/lox recombination, permanently labeling their progeny. We demonstrate that neural progenitors in neocortex generate discrete columnar structures that contain both projection neurons and protoplasmic astrocytes. Ninety-five percent of developmental cortical columns labeled in our system contained both astrocytes and neurons. The astrocyte to neuron ratio of labeled cells in a developmental column was 1:7.4, similar to the overall ratio of 1:8.4 across the entire gray matter of the neocortex, indicating that column-associated astrocytes account for the majority of protoplasmic astrocytes in neocortex. Most of the labeled columns contained multiple clusters of several astrocytes. Dividing cells were found at the base of neuronal columns at the beginning of gliogenesis, and later within the cortical layers, suggesting a mechanism by which astrocytes could be distributed within a column. These data indicate that radial glia are the source of both neurons and astrocytes in the neocortex, and that these two cell types are generated in a spatially restricted manner during cortical development

Evaluation and rational design of guide RNAs for efficient CRISPR/Cas9-mediated mutagenesis in Ciona

The CRISPR/Cas9 system has emerged as an important tool for various genome engineering applications. A current obstacle to high throughput applications of CRISPR/Cas9 is the imprecise prediction of highly active single guide RNAs (sgRNAs). We previously implemented the CRISPR/Cas9 system to induce tissue-specific mutations in the tunicate Ciona. In the present study, we designed and tested 83 single guide RNA (sgRNA) vectors targeting 23 genes expressed in the cardiopharyngeal progenitors and surrounding tissues of Ciona embryo. Using high-throughput sequencing of mutagenized alleles, we identified guide sequences that correlate with sgRNA mutagenesis activity and used this information for the rational design of all possible sgRNAs targeting the Ciona transcriptome. We also describe a one-step cloning-free protocol for the assembly of sgRNA expression cassettes. These cassettes can be directly electroporated as unpurified PCR products into Ciona embryos for sgRNA expression in vivo, resulting in high frequency of CRISPR/Cas9-mediated mutagenesis in somatic cells of electroporated embryos. We found a strong correlation between the frequency of an Ebf loss-of-function phenotype and the mutagenesis efficacies of individual Ebf-targeting sgRNAs tested using this method. We anticipate that our approach can be scaled up to systematically design and deliver highly efficient sgRNAs for the tissue-specific investigation of gene functions in Ciona

Rational design and whole-genome predictions of single guide RNAs for efficient CRISPR/Cas9-mediated genome editing in Ciona

The CRISPR/Cas9 system has emerged as an important tool for a wide variety of genome engineering applications, including reverse genetic screens. Previously, we described the implementation of the CRISPR/Cas9 system to induce tissue-specific mutations at targeted locations in the genome of the sea squirt Ciona (STOLFI et al. 2014). In the present study, we designed 83 single guide RNA (sgRNA) vectors targeting 23 genes expressed in the cardiopharyngeal progenitors and surrounding tissues in the Ciona embryo and measured their mutagenesis efficacy rates by massively parallel indel detection at the targeted loci using highthroughput sequencing. We show that the combined activity of two highly active sgRNAs allows us to generate large (>3 kbp) deletions of intervening genomic DNA in somatic cells of electroporated embryos, permitting tissue-specific gene knockouts. Additionally, we employed L1-regularized regression modeling to develop an optimal sgRNA design algorithm (TuniCUT), based on correlations between target sequence features and mutagenesis rates. Using this algorithm, we have predicted mutagenesis rates for sgRNAs targeting all 4,853,589 sites in the Ciona genome, which we have compiled into a "CRISPR/Cas9-induced Ciona Knock-Out" (Ci2KO) sgRNA sequence library. Finally, we describe a new method for the assembly of sgRNA expression cassettes using a simple one-step overlap PCR (OSO-PCR) protocol. These cassettes can be electroporated directly into Ciona embryos as unpurified PCR products to drive sgRNA expression, bypassing the need for time-consuming cloning and plasmid DNA preparations. We anticipate that this method will be used in combination with genome-wide sgRNA predictions to systematically investigate tissue-specific gene functions in Ciona

Genetic Labeling of Neuronal Subsets through Enhancer Trapping in Mice

The ability to label, visualize, and manipulate subsets of neurons is critical for elucidating the structure and function of individual cell types in the brain. Enhancer trapping has proved extremely useful for the genetic manipulation of selective cell types in Drosophila. We have developed an enhancer trap strategy in mammals by generating transgenic mice with lentiviral vectors carrying single-copy enhancer-detector probes encoding either the marker gene lacZ or Cre recombinase. This transgenic strategy allowed us to genetically identify a wide variety of neuronal subpopulations in distinct brain regions. Enhancer detection by lentiviral transgenesis could thus provide a complementary method for generating transgenic mouse libraries for the genetic labeling and manipulation of neuronal subsets

JohnsonEtAl-ACCs

From "Expression of smooth muscle-like effectors and core cardiomyocyte regulators in the contractile papillae of Ciona" Christopher J. Johnson, Florian Razy-Krajka, and Alberto Stolf

Regulation of neurogenesis by FGF signaling and Neurogenin in Ciona

Data and code from: "Regulation of neurogenesis by FGF signaling and Neurogenin in the invertebrate chordate Ciona" (2020) Kwantae Kim, Susanne Gibboney, Elijah K. Lowe, Wei Wang, & Alberto Stolfi Frontiers in Cell & Developmental Biology https://www.frontiersin.org/articles/10.3389/fcell.2020.00477/abstract PREPRINT: "Neurogenin regulates effectors of migratory neuron cell behaviors in Ciona" (2019) https://www.biorxiv.org/content/10.1101/654798v

Evolution and Development of a Minimal Nervous System in our Closest Invertebrate Relatives

Presented on November 25, 2019 at 11:15 a.m. in the Krone Engineered Biosystems Building, Room 1005.Alberto Stolfi is an Assistant Professor in the School of Biological Sciences at Georgia Tech. His research interests include neurodevelopment, neuroscience, developmental biology, cell biology, gene regulation, genome engineering, and tunicates.Runtime: 50:46 minutesAnimal behavior depends both on the intrinsic properties of individual neurons and how these neurons connect to and modulate one another. A major focus of modern neuroscience is to dissect behavior at the level of individual genes, neurons, and specific synaptic connections, but we are far from fully understanding how the composition and connectivity of even the smallest nervous systems can determine the wide range of behaviors observed in a free-living animal. Our lab is investigating the development of the simple larval nervous systems of tunicates like Ciona, marine invertebrates closely related to vertebrates. Although tunicates are chordates like us, Ciona larvae possess the smallest nervous system ever described at only 231 total neurons (177 central nervous system neurons and 54 peripheral sensory cells), comprising only the second complete “connectome” ever mapped. Using experimental tools such as CRISPR/Cas9-mediated mutagenesis and single-cell RNAseq, we have uncovered neurodevelopmental processes that shape this minimal nervous system, some of which are conserved even in mammals. We are also interested in studying an even more extreme example of the “minimization” of the tunicate nervous system, focusing on certain species that bypass the swimming larval phase and are therefore undergoing evolutionary loss of the larval nervous system altogether