CRISPR-Cas9 enables efficient sequence-specific mutagenesis for creating somatic or germline mutants of model organisms. Key constraints in vivo remain the expression and delivery of active Cas9-sgRNA ribonucleoprotein complexes (RNPs) with minimal toxicity, variable mutagenesis efficiencies depending on targeting sequence, and high mutation mosaicism. Here, we apply in vitro assembled, fluorescent Cas9-sgRNA RNPs in solubilizing salt solution to achieve maximal mutagenesis efficiency in zebrafish embryos. MiSeq-based sequence analysis of targeted loci in individual embryos using CrispRVariants, a customized software tool for mutagenesis quantification and visualization, reveals efficient biallelic mutagenesis that reaches saturation at several tested gene loci. Such virtually complete mutagenesis exposes loss-of-function phenotypes for candidate genes in somatic mutant embryos for subsequent generation of stable germline mutants. We further show that targeting of non-coding elements in gene regulatory regions using saturating mutagenesis uncovers functional control elements in transgenic reporters and endogenous genes in injected embryos. Our results establish that optimally solubilized, in vitro assembled fluorescent Cas9-sgRNA RNPs provide a reproducible reagent for direct and scalable loss-of-function studies and applications beyond zebrafish experiments that require maximal DNA cutting efficiency in vivo.

CRISPR-Cas9 and related technologies can efficiently alter genomic DNA at targeted positions and have far-reaching implications for functional screening and therapeutic gene editing. Understanding and unlocking this potential requires accurate evaluation of editing efficiency. Here, we show that methodological decisions for analyzing sequencing data can significantly affect mutagenesis efficiency estimates and we provide a comprehensive R-based toolkit, CrispRVariants and an accompanying web tool CrispRVariantsLite, that resolve and localize individual mutant alleles with respect to the endonuclease cut site. CrispRVariantsenabled analyses of newly generated and existing genome editing datasets underscore how careful consideration of the full variant spectrum gives insight toward effective guide and amplicon design as well as the mutagenic process.

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Cardiovascular lineages develop together with kidney, smooth muscle, and limb connective tissue progenitors from the lateral plate mesoderm (LPM). How the LPM initially emerges and how its downstream fates are molecularly interconnected remain unknown. Here, we isolate a pan-LPM enhancer in the zebrafish-specific draculin ( drl ) gene that provides specific LPM reporter activity from early gastrulation. In toto live imaging and lineage tracing of drl -based reporters captures the dynamic LPM emergence as lineage-restricted mesendoderm field. The drl pan-LPM enhancer responds to the transcription factors EomesoderminA, FoxH1, and MixL1 that combined with Smad activity drive LPM emergence. We uncover specific activity of zebrafish-derived drl reporters in LPM-corresponding territories of several chordates including chicken, axolotl, lamprey, Ciona , and amphioxus, revealing a universal upstream LPM program. Altogether, our work provides a mechanistic framework for LPM emergence as defined progenitor field, possibly representing an ancient mesodermal cell state that predates the primordial vertebrate embryo.

Much has been written in recent years about the changes in corporate strategies and industry structures associated with electronic coordination of market activities. This paper considers the advent of electronic market coordination in the home mortgage industry, focusing on Computerized Lean Origination (CL O) systems. Case studies of five CLOs (First Boston's Shelternet, PRC's LoanExpress, American Financial Network's Rennie Mae, Prudential's CLOS, and Citicorp's Mortgage Power Plus) reveal a range of system functionalities. Predictions from the Electronic Markets Hypothesis (EMH) are tested against the empirical results of the five case studies. As suggested by the EMH, financial intermediaries have been threatened by the in-"troduction of CLOS, and in some cases opposition has been mounted against the systems. On the other hand, despite the availability of the technology and mortgages' seemingly favorable characteristics as an electronically mediated market product, the industry has not been fundamentally changed by the introduction of these systems, despite more than a decade of experience with them. Of the two case studies that could be characterized as electronic markets, neither continues to exist in that form today. And the system with the largest dollar volume of mortgages of the five is best characterized as an electronic hierarchy. These results suggest that either the full results predicted by the EMH require a longer gestation period or that the underlying hypothesis will require augmentation in order to fully explain the results in the home mortgage market.