Analysis of off-target effects in CRISPR-based gene drives in the human malaria mosquito.

Autor:	Garrood WT; Department of Life Sciences, Imperial College London, SW7 2AZ London, United Kingdom., Kranjc N; Department of Life Sciences, Imperial College London, SW7 2AZ London, United Kingdom., Petri K; Molecular Pathology Unit, Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA 02129.; Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, MA 02129., Kim DY; Molecular Pathology Unit, Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA 02129.; Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, MA 02129., Guo JA; Molecular Pathology Unit, Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA 02129.; Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, MA 02129., Hammond AM; Department of Life Sciences, Imperial College London, SW7 2AZ London, United Kingdom.; W. Harry Feinstone Department of Molecular Microbiology and Immunology, The Johns Hopkins University, Baltimore, MD 21205., Morianou I; Department of Life Sciences, Imperial College London, SW7 2AZ London, United Kingdom., Pattanayak V; Molecular Pathology Unit, Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA 02129.; Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, MA 02129., Joung JK; Molecular Pathology Unit, Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA 02129.; Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, MA 02129., Crisanti A; Department of Life Sciences, Imperial College London, SW7 2AZ London, United Kingdom; a.drcrisanti@imperial.ac.uk a.simoni@pologgb.com.; Department of Molecular Medicine, University of Padova, 35121 Padova, Italy., Simoni A; Department of Life Sciences, Imperial College London, SW7 2AZ London, United Kingdom; a.drcrisanti@imperial.ac.uk a.simoni@pologgb.com.; Polo d'Innovazione Genomica, Genetica, e Biologia, 05100 Terni, Italy.
Jazyk:	angličtina
Zdroj:	Proceedings of the National Academy of Sciences of the United States of America [Proc Natl Acad Sci U S A] 2021 Jun 01; Vol. 118 (22). Date of Electronic Publication: 2021 Apr 30.
DOI:	10.1073/pnas.2004838117
Abstrakt:	CRISPR-Cas9 nuclease-based gene drives have been developed toward the aim of control of the human malaria vector Anopheles gambiae Gene drives are based on an active source of Cas9 nuclease in the germline that promotes super-Mendelian inheritance of the transgene by homology-directed repair ("homing"). Understanding whether CRISPR-induced off-target mutations are generated in Anopheles mosquitoes is an important aspect of risk assessment before any potential field release of this technology. We compared the frequencies and the propensity of off-target events to occur in four different gene-drive strains, including a deliberately promiscuous set-up, using a nongermline restricted promoter for SpCas9 and a guide RNA with many closely related sites (two or more mismatches) across the mosquito genome. Under this scenario we observed off-target mutations at frequencies no greater than 1.42%. We witnessed no evidence that CRISPR-induced off-target mutations were able to accumulate (or drive) in a mosquito population, despite multiple generations' exposure to the CRISPR-Cas9 nuclease construct. Furthermore, judicious design of the guide RNA used for homing of the CRISPR construct, combined with tight temporal constriction of Cas9 expression to the germline, rendered off-target mutations undetectable. The findings of this study represent an important milestone for the understanding and managing of CRISPR-Cas9 specificity in mosquitoes, and demonstrates that CRISPR off-target editing in the context of a mosquito gene drive can be reduced to minimal levels. Competing Interests: Competing interest statement: J.K.J. holds equity in Chroma Medicine and SeQure Dx, Inc. J.K.J. has financial interests in Beam Therapeutics, Editas Medicine, Excelsior Genomics, Pairwise Plants, Poseida Therapeutics, Transposagen Biopharmaceuticals, and Verve Therapeutics (formerly known as Endcadia). J.K.J.’s interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict-of-interest policies. K.P., V.P., and J.K.J. are coinventors on various patents and patent applications that describe gene editing and epigenetic editing technologies, including the CIRCLE-seq (circular in vitro reporting of cleavage effects by sequencing) assay used in this study.
Databáze:	MEDLINE
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