Menu

Close

Super-Mendelian inheritance mediated by CRISPR/Cas9 in the female mouse germline

Hannah A. Grunwald, Valentino M. Gantz, Gunnar Poplawski, Xiang-ru S. Xu, Ethan Bier, Kimberly L. Cooper

Preprint posted on July 07, 2018 https://www.biorxiv.org/content/early/2018/07/07/362558

A CRISPR-mediated Selfish Gene? Grunwald and colleagues design a “CopyCat” element that drives its own inheritance in the female mouse germline.

Selected by Rebekah Tillotson

Background

Modelling multigenic human diseases in mice can be very costly and time consuming due to the low frequency of animals that have the correct genotype. For example, if three independent alleles are involved, 146 offspring are required for a 90% chance of producing a single animal with the disease genotype. Recently, CRISPR/Cas9 technology has been employed to drive inheritance of specific alleles in insects. In these studies, a cassette consisting of Cas9 and a gRNA targeting a gene of interest was inserted into the site targeted by the gRNA, disrupting gene function. This cassette led to cleavage of the homologous allele, which when repaired by homology directed repair (HDR), copied the cassette into the second allele – thereby driving its own inheritance. Grunwald and colleagues investigate how this technology can be applied to mice.

Key findings

The authors designed a “CopyCat” element consisting of a gRNA targeting their gene of interest (Tyrosinase) and a fluorescent reporter (CMV-mCherry) – see figure below. This cassette was knocked-into exon 4 of the Tyrosinase gene, disrupting gene function. When bred with mice constitutively expressing Cas9, the gRNA and Cas9 protein are united at fertilisation in offspring. This resulted in homozygous loss-of-function, indicated by an albino phenotype. However, further analysis found that gene disruption was due to insertions/deletions (“indels”) resulting from non-homologous end joining (NHEJ), rather than homology directed repair (HDR) conversion using the “CopyCat” allele.

They hypothesised that mutagenesis was induced too late – missing the optimum window for HDR in early embryogenesis. They overcame this problem by controlling the activation of Cas9 expression with Cre recombinase. Activation of Cas9 during oogenesis with Vasa-Cre led to 45% of offspring in the next generation carrying the HDR converted allele (17% carrying indels and 38% where the gene was not mutated). While successful in the female germline, HDR was not detected when Cas9 was activated by either Vasa-Cre or Stra8-Cre in the male germline.

This system will dramatically improve disease model production in laboratory studies. While the high HDR efficiency achieved in the insect studies led to the proposal that this technology could reduce wild populations of malaria-carrying mosquitos, the authors conclude that this method should be optimised further before it could be considered applicable for the control of invasive rodent populations.

Figure 1: Schematic of the “CopyCat” system. (a) The “CopyCat” cassette was knocked into exon 4 of the Tyrosinase gene. (b) Cas9-mediated cleavage of the other allele can be repaired by homology directed repair (HDR), resulting in the copying of the “CopyCat” cassette and disruption of gene function.

 

What I like about this work

The authors have developed an elegant CRISPR-based system for driving inheritance of a modified allele. Their findings highlight the importance of tightly controlled Cas9 expression, furthering our understanding about the time windows when HDR is the predominant repair pathway in gametogenesis and early embryonic development. In contrast to the cassette used in insects, this is the first study that combines gRNA and Cas9 genes from different parents-of-origin. Altogether, it is an excellent example of how CRISPR/Cas9 technology can revolutionise in vivo studies.

Future directions/Comments/Questions for the authors

Ideally the method could be adapted to successfully drive inheritance in the male germline. The authors used existing Cas9 transgenic lines, limiting their choice to two constitutively expressed alleles that were activated by the removal of a “STOP” cassette by Cre drivers. The creation of novel transgenic lines where Cas9 is driven by cell-type specific promoters could limit Cas9 expression to the critical time window during spermatogenesis (and would simplify the method by eliminating the need for Cre transgenes). Promoters that transiently express Cas9 may also reduce toxicity – previously reported to occur when both Cas9 and gRNA are continually expressed. Lastly, I wonder whether the HDR rate would increase with a smaller cassette [the “CopyCat” cassette is 2.8 kb]?

Tags: crispr, gene drive, mouse

Posted on: 16th July 2018 , updated on: 17th July 2018

Read preprint (1 votes)




  • 1 comment

    9 months

    Kim Cooper

    Thank you for your preLight! I like your questions/future directions. Yes, we do think we can improve efficiency using a different promoter to drive Cas9 (and directly rather than in a conditional system). We have mice to work on that next step now. We are also interested in developing an inducible system once we get the timing right. Last, we aren’t sure if a smaller piece would mobilize more efficiently, but we are interested to see what happens to larger pieces.

    1

    Have your say

    Your email address will not be published. Required fields are marked *

    This site uses Akismet to reduce spam. Learn how your comment data is processed.

    Sign up to customise the site to your preferences and to receive alerts

    Register here

    Also in the genetics category:

    The Hunchback temporal transcription factor determines motor neuron axon and dendrite targeting in Drosophila

    Austin Q Seroka, Chris Q Doe



    Selected by Abagael Lasseigne

    1

    Preformed Chromatin Topology Assists Transcriptional Robustness of Shh during Limb Development

    Christina Paliou, Philine Guckelberger, Robert Schöpflin, et al.



    Selected by Rafael Galupa

    1

    Molecular Logic of Spinocerebellar Tract Neuron Diversity and Connectivity

    Myungin Baek, Vilas Menon, Thomas Jessell, et al.



    Selected by Yen-Chung Chen

    Over-activation of BMP signaling in neural crest cells precipitates heart outflow tract septation

    Jean Francois Darrigrand, Mariana Valente, Pauline Martinez, et al.



    Selected by Giulia Boezio

    Functional dissection of TADs reveals non-essential and instructive roles in regulating gene expression

    Alexandra Despang, Robert Schöpflin, Martin Franke, et al.



    Selected by Clarice Hong

    Crowdfunded whole-genome sequencing of the celebrity cat Lil BUB identifies causal mutations for her osteopetrosis and polydactyly

    Mike Bridavsky, Heiner Kuhl, Arthur Woodruf, et al.



    Selected by Jesus Victorino, Gabriel Aughey

    1

    Dynamic Erasure of Random X-Chromosome Inactivation during iPSC Reprogramming

    Adrian Janiszewski, Irene Talon, Juan Song, et al.



    Selected by Sergio Menchero

    Kinesin-6 Klp9 plays motor-dependent and -independent roles in collaboration with Kinesin-5 Cut7 and the microtubule crosslinker Ase1 in fission yeast

    Masashi Yukawa, Masaki Okazaki, Yasuhiro Teratani, et al.



    Selected by I. Bouhlel

    Human Handedness: Genetics, Microtubules, Neuropsychiatric Diseases and Brain Language Areas

    Akira Wiberg, Gwenaelle Douaud, Michael Ng, et al.



    Selected by Jose Guerrero

    The Spatio-Temporal Control of Zygotic Genome Activation

    George Gentsch, Nick D. L. Owens, James C. Smith



    Selected by Meng Zhu

    Multilevel regulation of the glass locus during Drosophila eye development

    Cornelia Fritsch, F. Javier Bernardo-Garcia, Tim Humberg, et al.



    Selected by Gabriel Aughey

    1

    Distinct ROPGEFs successively drive polarization and outgrowth of root hairs

    Philipp Denninger, Anna Reichelt, Vanessa Aphaia Fiona Schmidt, et al.



    Selected by Marc Somssich

    A direct and widespread role for the nuclear receptor EcR in mediating the response to ecdysone in Drosophila

    Christopher M Uyehara, Daniel J McKay



    Selected by Natalie Dye

    MRE11-RAD50-NBS1 activates Fanconi Anemia R-loop suppression at transcription-replication conflicts

    Emily Yun-Chia Chang, James P Wells, Shu-Huei Tsai, et al.



    Selected by Katie Weiner

    1

    Super-resolution Molecular Map of Basal Foot Reveals Novel Cilium in Airway Multiciliated Cells

    Quynh Nguyen, Zhen Liu, Rashmi Nanjundappa, et al.



    Selected by Robert Mahen

    Single cell RNA-Seq reveals distinct stem cell populations that drive sensory hair cell regeneration in response to loss of Fgf and Notch signaling

    Mark E. Lush, Daniel C. Diaz, Nina Koenecke, et al.

    AND

    Distinct progenitor populations mediate regeneration in the zebrafish lateral line.

    Eric D Thomas, David Raible



    Selected by Rudra Nayan Das

    2

    Also in the molecular biology category:

    The autophagic membrane tether ATG2A transfers lipids between membranes

    Shintaro Maeda, Chinatsu Otomo, Takanori Otomo



    Selected by Sandra Malmgren Hill

    LTK is an ER-resident receptor tyrosine kinase that regulates secretion

    Federica G. Centonze, Veronika Reiterer, Karsten Nalbach, et al.



    Selected by Nicola Stevenson

    1

    Accurate detection of m6A RNA modifications in native RNA sequences

    Huanle Liu, Oguzhan Begik, Morghan C Lucas, et al.



    Selected by Christian Bates

    1

    Blue light induces neuronal-activity-regulated gene expression in the absence of optogenetic proteins

    Kelsey M. Tyssowski, Jesse M. Gray



    Selected by Zheng-Shan Chong

    Slide-seq: A Scalable Technology for Measuring Genome-Wide Expression at High Spatial Resolution

    Samuel G Rodriques, Robert R Stickels, Aleksandrina Goeva, et al.

    AND

    High-density spatial transcriptomics arrays for in situ tissue profiling

    Sanja Vickovic, Goekcen Eraslan, Johanna Klughammer, et al.



    Selected by Carmen Adriaens

    Optical determination of absolute membrane potential

    Julia R. Lazzari-Dean, Anneliese M.M. Gest, Evan Miller



    Selected by James Marchant

    MicroRNA-mediated control of developmental lymphangiogenesis

    Hyun Min Jung, Ciara Hu, Alexandra M Fister, et al.



    Selected by Rudra Nayan Das

    Microfluidic protein isolation and sample preparation for high resolution cryo-EM

    Claudio Schmidli, Stefan Albiez, Luca Rima, et al.



    Selected by David Wright

    A DNA-based voltmeter for organelles

    Anand Saminathan, John Devany, Kavya S Pillai, et al.



    Selected by Robert Mahen

    1

    Structures of the Otopetrin Proton Channels Otop1 and Otop3

    Kei Saotome, Bochuan Teng, Che Chun (Alex) Tsui, et al.



    Selected by David Wright

    Central spindle microtubules are strongly coupled to chromosomes during both anaphase A and anaphase B

    Che-Hang Yu, Stefanie Redemann, Hai-Yin Wu, et al.



    Selected by Federico Pelisch

    1

    Cell growth dilutes the cell cycle inhibitor Rb to trigger cell division

    Evgeny Zatulovskiy, Daniel F. Berenson, Benjamin R. Topacio, et al.



    Selected by Zaki Ahmad

    1

    Distinct ROPGEFs successively drive polarization and outgrowth of root hairs

    Philipp Denninger, Anna Reichelt, Vanessa Aphaia Fiona Schmidt, et al.



    Selected by Marc Somssich

    Inactive USP14 and inactive UCHL5 cause accumulation of distinct ubiquitinated proteins in mammalian cells

    Jayashree Chadchankar, Victoria Korboukh, Peter Doig, et al.



    Selected by Mila Basic

    Bacteriophage resistance alters antibiotic mediated intestinal expansion of enterococci

    Anushila Chatterjee, Cydney N Johnson, Phat Luong, et al.



    Selected by Yasmin Lau

    On-site ribosome remodeling by locally synthesized ribosomal proteins in axons

    Toshiaki Shigeoka, Max Koppers, Hovy Ho-Wai Wong, et al.



    Selected by Srivats Venkataramanan

    Also in the synthetic biology category:

    The autophagic membrane tether ATG2A transfers lipids between membranes

    Shintaro Maeda, Chinatsu Otomo, Takanori Otomo



    Selected by Sandra Malmgren Hill

    Engineered Enzymes that Retain and Regenerate their Cofactors Enable Continuous-Flow Biocatalysis

    Carol J. Hartley, Charlotte C. Williams, Judith A. Scoble, et al.



    Selected by Zhang-He Goh

    1

    Blue light induces neuronal-activity-regulated gene expression in the absence of optogenetic proteins

    Kelsey M. Tyssowski, Jesse M. Gray



    Selected by Zheng-Shan Chong

    Aqueous synthesis of a small-molecule lanthanide chelator amenable to copper-free click chemistry

    Stephanie Cara Bishop, Robert Winefield, Asokan Anbanandam, et al.



    Selected by Zhang-He Goh

    Synthetic pluripotent bacterial stem cells

    Sara Molinari, David L. Shis, James Chappell, et al.



    Selected by Lorenzo Lafranchi

    A DNA-based voltmeter for organelles

    Anand Saminathan, John Devany, Kavya S Pillai, et al.



    Selected by Robert Mahen

    1

    Variability of bacterial behavior in the mammalian gut captured using a growth-linked single-cell synthetic gene oscillator

    David T Riglar, David L Richmond, Laurent Potvin-Trottier, et al.



    Selected by Meng Zhu

    A direct selection strategy for isolating aptamers with pH-sensitive binding activity

    Chelsea K L Gordon, Michael Eisenstein, H T Soh



    Selected by Zhang-He Goh

    SABER enables highly multiplexed and amplified detection of DNA and RNA in cells and tissues

    Jocelyn Y. Kishi, Brian J. Beliveau, Sylvain W. Lapan, et al.



    Selected by Yen-Chung Chen

    LADL: Light-activated dynamic looping for endogenous gene expression control

    Mayuri Rege, Ji Hun Kim, Jacqueline Valeri, et al.



    Selected by Ivan Candido-Ferreira

    Glutamic acid is a carrier for hydrazine during the biosyntheses of fosfazinomycin and kinamycin

    Kwo-Kwang Abraham Wang, Tai L. Ng, Peng Wang, et al.



    Selected by Ellis O'Neill

    Super-Mendelian inheritance mediated by CRISPR/Cas9 in the female mouse germline

    Hannah A. Grunwald, Valentino M. Gantz, Gunnar Poplawski, et al.



    Selected by Rebekah Tillotson

    1

    Controlling CRISPR-Cas9 with ligand-activated and ligand-deactivated sgRNAs

    Kale Kundert, James E Lucas, Kyle E Watters, et al.



    Selected by Samantha Seah
    Close