@article{da04bdcbf65c4808b51040dbb5e876e0,
title = "Cas9-mediated gene editing in the black-legged tick, Ixodes scapularis, by embryo injection and ReMOT Control",
abstract = "Despite their capacity to acquire and pass on an array of debilitating pathogens, research on ticks has lagged behind other arthropod vectors, such as mosquitoes, largely because of challenges in applying available genetic and molecular tools. CRISPR-Cas9 is transforming non-model organism research; however, successful gene editing has not yet been reported in ticks. Technical challenges for injecting tick embryos to attempt gene editing have further slowed research progress. Currently, no embryo injection protocol exists for any chelicerate species, including ticks. Herein, we report a successful embryo injection protocol for the black-legged tick, Ixodes scapularis, and the use of this protocol for genome editing with CRISPR-Cas9. We also demonstrate that the ReMOT Control technique could be successfully used to generate genome mutations outside Insecta. Our results provide innovative tools to the tick research community that are essential for advancing our understanding of the molecular mechanisms governing pathogen transmission by tick vectors and the underlying biology of host-vector-pathogen interactions.",
author = "Arvind Sharma and Pham, {Michael N.} and Reyes, {Jeremiah B.} and Randeep Chana and Yim, {Won C.} and Heu, {Chan C.} and Donghun Kim and Duverney Chaverra-Rodriguez and Rasgon, {Jason L.} and Harrell, {Robert A.} and Nuss, {Andrew B.} and Monika Gulia-Nuss",
note = "Funding Information: We thank Prof. Timothy Kurtti for sharing the CMV/CAGGS plasmid. We acknowledge Channa-Aluvihare and Yonas Gebremicale, Insect Transformation Center, for their insight during the initial phase of embryo injection protocol development. We also thank Drs. Juli Petereit and Hans Vasquez-Gross, the Nevada Bioinformatics Center, for deep sequencing data analysis. This project was funded by NIH - NIAID R21AI128393 and Plymouth Hill Foundation , NY to MG-N, Startup funds from the University of Nevada to AN, Peer-to-Peer Grant from IGTRCN to AS, NIH - NIAID R21AI111175 , USDA - NIFA 2014-10320 , USDA Hatch funds (Project # PEN04769 ), and NSF - BIO 1645331 to JLR. This work was in part supported by the NV INBRE ( GM103440 ) and MW CTR -IN ( 5 U54 GM104944 ) grant from the National Institute of General Medical Sciences from the National Institutes of Health (NIH). Funding Information: We thank Prof. Timothy Kurtti for sharing the CMV/CAGGS plasmid. We acknowledge Channa-Aluvihare and Yonas Gebremicale, Insect Transformation Center, for their insight during the initial phase of embryo injection protocol development. We also thank Drs. Juli Petereit and Hans Vasquez-Gross, the Nevada Bioinformatics Center, for deep sequencing data analysis. This project was funded by NIH-NIAID R21AI128393 and Plymouth Hill Foundation, NY to MG-N, Startup funds from the University of Nevada to AN, Peer-to-Peer Grant from IGTRCN to AS, NIH-NIAID R21AI111175, USDA-NIFA 2014-10320, USDA Hatch funds (Project #PEN04769), and NSF-BIO 1645331 to JLR. This work was in part supported by the NV INBRE (GM103440) and MW CTR-IN (5 U54 GM104944) grant from the National Institute of General Medical Sciences from the National Institutes of Health (NIH). MG-N, ABN, JLR, and RH conceived and designed the experiments. ABN provided technical and intellectual support. AS developed an embryo injection protocol and carried out CRISPR embryo injections. JR assisted in screening the larvae hatched from the injected eggs, DNA extraction, and sequencing. MP developed the ReMOT Control adult injection protocol in consultation with DCR, DK, and JLR, and screened larvae hatched from the adult injections. RC (with MP) optimized the Chelex DNA extraction protocol and assisted in DNA extractions. WCY assembled the new tick genome and identified full-length sequences of genes. DCR, DK, CCH, and JLR generated ReMOT Control reagents. AS and MP wrote the initial drafts of the manuscript. MG-N, ABN, AS, MP, and JLR wrote the final draft. MG-N, ABN, AS, and MP revised the manuscript. JLR, DCR, and CCH have filed for patent protection on the ReMOT Control technology. No other authors have any competing interests. Publisher Copyright: {\textcopyright} 2022 The Authors",
year = "2022",
month = mar,
day = "18",
doi = "10.1016/j.isci.2022.103781",
language = "English (US)",
volume = "25",
journal = "iScience",
issn = "2589-0042",
publisher = "Elsevier Inc.",
number = "3",
}