Simultaneous paralogue knockout using a CRISPR-concatemer in mouse small intestinal organoids

Approaches based on genetic modification have been invaluable for investigating a wide array of biological processes, with gain- and loss-of-function approaches frequently used to investigate gene function. However, the presence of paralogues, and hence possible genetic compensation, for many genes necessitates the knockout (KO) of all paralogous genes in order to observe clear phenotypic change. CRISPR technology, the most recently described tool for gene editing, can generate KOs with unprecedented ease and speed and has been used in adult stem cell-derived organoids for single gene knockout, gene knock-in and gene correction. However, the simultaneous targeting of multiple genes in organoids by CRISPR technology has not previously been described. Here we describe a rapid, scalable and cost effective method for generating double knockouts in organoids. By concatemerizing multiple gRNA expression cassettes, we generated a ‘gRNA concatemer vector’. Our method allows the rapid assembly of annealed synthetic DNA oligos into the final vector in a single step. This approach facilitates simultaneous delivery of multiple gRNAs to allow up to 4 gene KO in one step, or potentially to increase the efficiency of gene knockout by providing multiple gRNAs targeting one gene. As a proof of concept, we knocked out negative regulators of the Wnt pathway in small intestinal organoids, thereby removing their growth dependence on the exogenous Wnt enhancer, R-spondin1.A.A-R. is supported by the Medical Research Council (MRC), A.M.is supported by Wntsapp (Marie Curie ITN) and B-K.K. and R.M. are supported by a Sir Henry Dale Fellowship from the Wellcome Trust and the Royal Society [101241/Z/13/Z] and receive support through a core grant from the Wellcome Trust and MRC to the WT-MRC Cambridge Stem Cell Institute

A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer

This protocol describes the steps for cloning multiple single guide RNAs into one guide RNA concatemer vector, which is of particular use in creating multi-gene knockouts using CRISPR/Cas9 technology. The generation of double knockouts in intestinal organoids is shown as a possible application of this method.A.M. is supported by Wntsapp (Marie Curie ITN), A.A-R. is supported by the Medical Research Council (MRC), and B-K.K. and R.M. are supported by a Sir Henry Dale Fellowship from the Wellcome Trust and the Royal Society [101241/Z/13/Z] and receive support through a core grant from the Wellcome Trust and MRC to the Wellcome Trust - MRC Cambridge Stem Cell Institute

LGR6 Is a High Affinity Receptor of R-Spondins and Potentially Functions as a Tumor Suppressor

BACKGROUND: LGR6 (leucine-rich repeat containing, G protein-coupled receptor 6) is a member of the rhodopsin-like seven transmembrane domain receptor superfamily with the highest homology to LGR4 and LGR5. LGR6 was found as one of the novel genes mutated in colon cancer through total exon sequencing and its promoter region is hypermethylated in 20-50% of colon cancer cases. In the skin, LGR6 marks a population of stem cells that can give rise to all cell lineages. Recently, we and others demonstrated that LGR4 and LGR5 function as receptors of R-spondins to potentiate Wnt/β-catenin signaling. However, the binding affinity and functional response of LGR6 to R-spondins, and the activity of colon cancer mutants of LGR6 have not been determined. PRINCIPAL FINDINGS: We found that LGR6 also binds and responds to R-spondins 1-3 with high affinity to enhance Wnt/β-catenin signaling through increased LRP6 phosphorylation. Similar to LGR4 and LGR5, LGR6 is not coupled to heterotrimeric G proteins or to β-arrestin following R-spondin stimulation. Functional and expression analysis of three somatic mutations identified in colon cancer samples indicates that one mutant fails to bind and respond to R-spondin (loss-of-function), but the other two have no significant effect on receptor function. Overexpression of wild-type LGR6 in HeLa cells leads to increased cell migration following co-treatment with R-spondin1 and Wnt3a when compared to vector control cells or cells overexpressing the loss-of-function mutant. CONCLUSIONS: LGR6 is a high affinity receptor for R-spondins 1-3 and potentially functions as a tumor suppressor despite its positive effect on Wnt/β-catenin signaling

Primary Intestinal Epithelial Organoid Culture

The intestinal epithelium is known as one of the most regenerative tissues in our body. The lining of the intestine is composed of a single layer of epithelial cells generated by rapidly renewing stem cells residing at the crypt bottoms, resulting in a flow of cells to the villus tips. The stereotypical crypt-villus architecture makes the intestine an ideal model for stem cell research. Based on recent advances in research of stem cell niche signals in vivo, we have established an intestinal epithelial stem cell culture method. Under this culture condition, single Lgr5+ intestinal stem cells (ISCs) or isolated whole crypts efficiently expand into three-dimensional spherical structures recapitulating the intestinal crypt-villus organization. These organoids can be passaged weekly and maintained for years in culture. Moreover, they can be cryopreserved. As intestinal organoids recapitulate many aspects of the epithelial biology and are amenable to most, if not all, current experimental manipulations, they are widely used to study stem cell biology, cell fate determination, gene function, and disease mechanism

A novel culture system for adult porcine intestinal crypts

BACKGROUND: Porcine models are useful for investigating therapeutic approaches to short bowel syndrome and potentially to intestinal stem cell (ISC) transplantation. While techniques for the culture and genetic manipulation of ISCs from mice and humans are well established, similar methods for porcine stem cells have not been reported. METHODS: Jejunal crypts were isolated from murine, human, and juvenile and adult porcine small intestine, suspended in Matrigel, and co-cultured with syngeneic intestinal subepithelial myofibroblasts (ISEMFs) or cultured without feeder cells in various culture media. Media containing epidermal growth factor, noggin, and R-spondin 1 (ENR medium) were supplemented with various combinations of Wnt3a- or ISEMF-conditioned medium (CM), and glycogen synthase kinase 3 inhibitor (GSK3i) and studied on cultured crypts. Cell lineage differentiation was assessed by immunohistochemistry (IHC) and quantitative PCR (qPCR). Cultured porcine cells were serially passaged and transduced with a lentiviral vector. RESULTS: Whereas ENR medium supported murine enteroid growth; it did not sustain porcine crypts beyond 5 days. Supplementation of Wnt3a-CM and GSK3i resulted in the formation of complex porcine enteroids with budding extensions. These enteroids contained a mixture of stem and differentiated cells and were successfully passaged in the presence of GSK3i. Crypts grown in media supplemented with porcine ISEMF-CM formed spheroids that were less well differentiated than enteroids. Enteroids and spheroids were transfected with a lentivirus with high efficiency. DISCUSSION: We describe a method to maintain juvenile and adult porcine crypt cells long-term in culture. Porcine enteroids and spheroids can be successfully passaged and transduced using lentiviral vectors

Clinical Electromyographycal Study on Gamma System -Selective Gamma fibere Blocking with Procaine Anesthesia-

It was observed in the preliminary study that there was diminution of the H reflex in the calf muscles with hypoactive ankle jerk after gamma fibre blocking with procaine injected in the tibial nerve. Although no remarkable change on motor power of the muscle was seen. The present investigation deals with mechanism of this diminution of the H reflex and with regulation of motor unit discharge during voluntary contraction after gamma fiber blocking. 1. Diminution of the H reflex. Amplitude of H reflex response which had been diminished after gamma fibere blocking was recovered by voluntary contraction of the calf muscles. There was little change observed in the latent period of the H reflex between the experimental states with and without voluntary contraction. Excitability of the motoneurone measured by the H reflex recovery curve with conditioning and test stimulus indicated that there was a marked increase of excitability of the motoneurone during voluntary contraction of the muscles. These results suggest that the diminution of the H reflex after gamma fibre blocking is due to decreased motoneurone excitability which is a result of diminished afferent impulse to the motoneurone from the spindle. On the other hand, augmentation of the H reflex response during voluntary contraction is due to increased motoneurone excitability where descending impulse to the motonerone from upper motor neurone compensate the loss of afferent impulse from the spindle. 2. Effect of gamma blocking upon veluntary contraction. There was never observed the démblée type of discharge at the onset of contraction and could not do a quick contraction of the muscle. There was seen prolonged reaction time on the E. M. G. although no decrease of motor power detected after gamma bloking. This may indicate again that there is decrease of motoneurone excitability after gamma blocking and it takes a little time to recover this decreased excitability by impulse from upper motor neurone at the onset of voluntary contraction