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CRISPR gRNA Resources

Resource	Description	Author/Source
CCTop	CRISPR/Cas9 Target online predictor. Support for multiple species including Drosophila (release 5), zebrafish, mouse, human, and others. Notably large set of configurable parameters.	Center for Organismal Studies University of Heidelberg Heidelberg, Germany
CRISPR Optimal Target Finder	Identifies gRNA targets within a provided sequence and additionally searches genome-wide (release 6, current FlyBase release) for potential off-target sites. Includes several Drosophila species.	O'Connor-Giles, Wildonger, and Harrison Labs University of Wisconsin-Madison WI, USA
CRISPRscan	Searchable and browsable collection of genome-wide (release 6, current FlyBase release) sgRNA target sites. Displays sgRNA sites as tracks on UCSC's genome browser and allows searching via gene or prediction via sequence. Support for multiple species (fly, zebrafish, mouse, human, others).	Giraldez Lab Yale University New Haven, CT, USA
DGRC sgRNA libraries	Guide RNA collections available from the Drosophila Genomics Resource Center (DGRC)	Indiana University Bloomington, IN, USA
DRSC Find CRISPRs Tool	A tool for discovering gRNA targets located throughout the fly genome (release 6, current FlyBase release). Searchable by gene ID, symbol, or chromosome location. Includes options to modify mismatch stringency for off-targets.	Drosophila RNAi Screening Center (DRSC) Harvard Medical School Boston, MA, USA
E-CRISP	gRNA genome-wide target finder, searchable via gene symbol or sequence. Relaxed, "medium", or strict search options. Support for multiple species including Drosophila (release 5), zebrafish, mouse, human, and others.	Boutros Lab, DKFZ Heidelberg, Germany
PlatinumCRISPr	gRNA target finder. Incorporates correct sgRNA folding, various sequence elements and sgRNA/Cas9 structural features to predict high cleavage efficiency. Also allows separate crRNA and tracrRNA folding analysis. Applicable to any organism.	Soller Lab, School of Biosciences Arnold Lab, Institute of Cancer and Genome Sciences University of Birmingham Birmingham, UK

CRISPR Stocks and Cell Lines

Resource	Description	Source/Reference
Stocks for Genome Editing	Stocks for expression of both Cas9 nuclease and tracrRNA, Guide RNA Stocks (includes the TRiP collections), and CRISPR - UAS-Cas9 & GAL4 (includes the TRIP-CRISPR Toolbox set)}.	Bloomington Drosophila Stock Center (BDSC) Indiana University Bloomington, IN, USA
Heidelberg CFD CRISPR Library Stocks	This library, for tissue specific CRISPR mutagenesis, was created by the laboratory of Michael Boutros at the DKFZ (see CRISPR fly design) and donated to the VDRC for distribution	DKFZ Heidelberg, Germany
NIG-FLY CRISPR/Cas9	Reagents, protocols, and online tools developed for genome engineering in Drosophila.	NIG-FLY National Institute of Genetics Mishima, Japan
DGRC CRISPR Cell Lines	A catalog of CRISPR Cell lines at the Drosophila Genomics Resource Center (DGRC)	Indiana University Bloomington, IN, USA

CRISPR Vectors

Resource	Description	Author/Source
Addgene CRISPR/Cas9 Plasmids for use in insects	A list of available Cas9 & gRNA expression plasmids for use in insects, including Drosophila.	Addgene Cambridge, MA, USA
CRISPR Fly Design Plasmids	Descriptions and information regarding the plasmids created by the CRISPR Fly Design group (also deposited at Addgene).	MRC Laboratory of Molecular Biology Cambridge, UK
DGRC CRISPR Vectors	A catalog of useful CRISPR vectors available from the Drosophila Genomics Resource Center (DGRC)	Inidana University Bloomington, IN, USA
NIG-FLY CRISPR/Cas9	Reagents, protocols, and online tools developed for genome engineering in Drosophila.	NIG-FLY National Institute of Genetics Mishima, Japan

Additional Useful CRISPR Links

Resource	Description	Author/Source
CRISPRESSO2	Analysis of genome editing outcomes from deep sequencing data	Broad Institute, Massachusetts General Hospital, Harvard Medical School, Children's Hospital, Dana Farber Cancer Institute Cambridge, MA Boston, MA
CRISPR Fly Design	Reagents, protocols, and results from fly CRISPR/Cas9 experiments.	MRC Laboratory of Molecular Biology Cambridge, UK
flyCRISPR	An overview and resource collection for CRISPR gene editing in Drosophila.	O'Connor-Giles, Wildonger, and Harrison Labs University of Wisconsin-Madison WI, USA
DRSC cell-based CRISPR modification	Useful references for cell-based CRISPR.	Drosophila RNAi Screening Center (DRSC) Harvard Medical School Boston, MA, USA
TRiP CRISPR fly stock information	Links and descriptions for TRiP-CRISPR overexpression stocks, TRiP-CRISPR knockout stocks, and TRiP-CRISPR toolbox stocks	Drosophila RNAi Screening Center (DRSC) Harvard Medical School Boston, MA, USA

Selected CRISPR method reviews

• Venken KJ, Sarrion-Perdigones A, Vandeventer PJ, Abel NS, Christiansen AE, Hoffman KL. Genome engineering: Drosophila melanogaster and beyond. Wiley Interdiscip Rev Dev Biol. 2015 Oct 8. doi: 10.1002/wdev.214. [Epub ahead of print] Review. PubMed PMID: 26447401.
• Gratz1 SJ, Rubinstein CD, Harrison MM, Wildonger J, O'Connor-Giles KM CRISPR-Cas9 Genome Editing in Drosophila Curr Protoc Mol Biol. 2015 Jul 1;111:31.2.1-31.2.20. doi: 10.1002/0471142727.mb3102s111. PMID: 26131852; PMCID: PMC4506758.
• Beumer KJ, Carroll D. Targeted genome engineering techniques in Drosophila. Methods. 2014 Jun 15;68(1):29-37. doi: 10.1016/j.ymeth.2013.12.002. Epub 2014 Jan 8. Review. PubMed PMID: 24412316; PubMed Central PMCID: PMC4048800.
• Xu J, Ren X, Sun J, Wang X, Qiao HH, Xu BW, Liu LP, Ni JQ. A Toolkit of CRISPR-Based Genome Editing Systems in Drosophila. J Genet Genomics. 2015 Apr 20;42(4):141-9. doi: 10.1016/j.jgg.2015.02.007. Epub 2015 Mar 12. Review. PubMed PMID: 25953352.

Selected CRISPR method reports for tissue culture cells

• Bassett AR, Tibbit C, Ponting CP, Liu JL. Mutagenesis and homologous recombination in Drosophila cell lines using CRISPR/Cas9. Biol Open. 2014 Jan 15;3(1):42-9. doi: 10.1242/bio.20137120. PMID: 24326186

• Bassett AR, Kong L, Liu JL. A genome-wide CRISPR library for high-throughput genetic screening in Drosophila cells. J Genet Genomics. 2015 Jun 20;42(6):301-9. doi: 10.1016/j.jgg.2015.03.011. Epub 2015 Apr 18. PMID: 26165496

• Bosch JA, Colbeth, R, Zirin, J, Perrimon, N. Gene Knock-Ins in Drosophila Using Homology-Independent Insertion of Universal Donor Plasmids.. Genetics 214(1): 75--89. doi: 10.1534/genetics.119.302819. PMID: 31685521

• Bosch JA, Knight S, Kanca O, Zirin J, Yang-Zhou D, Hu Y, Rodiger J, Amador G, Bellen HJ, Perrimon N. Use of the CRISPR-Cas9 System in Drosophila Cultured Cells to Introduce Fluorescent Tags into Endogenous Genes Curr Protoc Mol Biol. 2020 Mar;130(1):e112. doi: 10.1002/cpmb.112. PMID: 31869524

• Böttcher R, Hollmann M, Merk K, Nitschko V, Obermaier C, Philippou-Massier J, Wieland I, Gaul U, Förstemann K. Efficient chromosomal gene modification with CRISPR/cas9 and PCR-based homologous recombination donors in cultured Drosophila cells. Nucleic Acids Res. 2014 Jun;42(11):e89. doi: 10.1093/nar/gku289. Epub 2014 Apr 19. PMID: 24748663

• Housden BE, Valvezan AJ, Kelley C, Sopko R, Hu Y, Roesel C, Lin S, Buckner M, Tao R, Yilmazel B, Mohr SE, Manning BD, Perrimon N. Identification of potential drug targets for tuberous sclerosis complex by synthetic screens combining CRISPR-based knockouts with RNAi. Sci Signal. 2015 Sep 8;8(393):rs9. doi: 10.1126/scisignal.aab3729. PMID: 26350902

• Huynh N, Depner N, Larson R, King-Jones K. A versatile toolkit for CRISPR-Cas13-based RNA manipulation in Drosophila bioRxiv. doi: https://doi.org/10.1101/2020.09.25.314047

• Kunzelmann S, Böttcher R, Schmidts I, Förstemann K. A comprehensive toolbox for genome editing in cultured Drosophila cells. G3 (Bethesda). 2016 Apr 13; doi:10.1534/g3.116.028241

• Viswanatha R, Brathwaite R, Hu Y, Li Z, Rodiger J, Merckaert P, Chung V, Mohr SE, Perrimon N. Pooled CRISPR Screens in Drosophila Cells Curr Protoc Mol Biol. 2019 Dec;129(1):e111. doi: 10.1002/cpmb.111. PMID: 31763777

• Viswanatha, R, Zaffagni, M, Zirin, J, Perrimon, N, Kadener, S. CRISPR-Cas13 mediated Knock Down in Drosophila cultured cells bioRxiv. doi: https://doi.org/10.1101/2020.11.01.364166

• Xia, B, Amador, G, Viswanatha, R, Zirin, J, Mohr, SE, Perrimon, N. (2020). CRISPR-based engineering of gene knockout cells by homology-directed insertion in polyploid Drosophila S2R+ cells. Nat. Protoc., 21 Sept. 2020 15(10): 3478-3498. doi:10.1038/s41596-020-0383-8 PubMed PMID: 32958931

Selected CRISPR method reports for in vivo

• Bassett AR, Tibbit C, Ponting CP, Liu JL. Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system. Cell Rep. 2013 Jul 11;4(1):220-8. doi: 10.1016/j.celrep.2013.06.020. Epub 2013 Jul 1. PMID: 23827738

• Bosch JA, Colbeth, R, Zirin, J, Perrimon, N. Gene Knock-Ins in Drosophila Using Homology-Independent Insertion of Universal Donor Plasmids.. Genetics 214(1): 75--89. doi: 10.1534/genetics.119.302819. PMID: 31685521

• Bosch JA, Birchak, G, Perrimon, N. Precise genome engineering in Drosophila using prime editing. Proc. Natl. Acad. Sci. U.S.A. 118(1): e2021996118. doi: 10.1073/pnas.2021996118. PMID: 33443210

• Gratz SJ, Cummings AM, Nguyen JN, Hamm DC, Donohue LK, Harrison MM, Wildonger J, O'Connor-Giles KM. Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease. Genetics. 2013 Aug;194(4):1029-35. doi: 10.1534/genetics.113.152710. Epub 2013 May 24. PMID: 23709638

• Huynh N, Depner N, Larson R, King-Jones K. A versatile toolkit for CRISPR-Cas13-based RNA manipulation in Drosophila bioRxiv. doi: https://doi.org/10.1101/2020.09.25.314047.

• Kanca O, Zirin J, Garcia-Marques J, Knight SM, Yang-Zhou D, Amador G, Chung H, Zuo Z, Ma L, He Y, Lin WW, Fang Y, Ge M, Yamamoto S, Schulze K, Hu Y, Spradling AC, Mohr SE, Perrimon N, Bellen HJ. An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms Elife. 2019 Nov 1;8:e51539. doi: 10.7554/eLife.51539. PMID: 31674908

• Kondo S, Ueda R. Highly improved gene targeting by germline-specific Cas9 expression in Drosophila. Genetics. 2013 Nov;195(3):715-21. doi: 10.1534/genetics.113.156737. Epub 2013 Sep 3. PMID: 24002648

• Port F, Muschalik N, Bullock SL. Systematic Evaluation of Drosophila CRISPR Tools Reveals Safe and Robust Alternatives to Autonomous Gene Drives in Basic Research. G3 (Bethesda). 2015 May 20;5(7):1493-502. doi: 10.1534/g3.115.019083. PMID: 25999583

• Ren X, Sun J, Housden BE, Hu Y, Roesel C, Lin S, Liu LP, Yang Z, Mao D, Sun L, Wu Q, Ji JY, Xi J, Mohr SE, Xu J, Perrimon N, Ni JQ. Optimized gene editing technology for Drosophila melanogaster using germ line-specific Cas9. Proc Natl Acad Sci U S A. 2013 Nov 19;110(47):19012-7. doi: 10.1073/pnas.1318481110. Epub 2013 Nov 4. PMID: 24191015

• Ren X, Yang Z, Xu J, Sun J, Mao D, Hu Y, Yang SJ, Qiao HH, Wang X, Hu Q, Deng P, Liu LP, Ji JY, Li JB, Ni JQ. Enhanced specificity and efficiency of the CRISPR/Cas9 system with optimized sgRNA parameters in Drosophila. Cell Rep. 2014 Nov 6;9(3):1151-62. doi: 10.1016/j.celrep.2014.09.044. Epub 2014 Oct 23. PMID: 25437567

• Viswanatha, R, Zaffagni, M, Zirin, J, Perrimon, N, Kadener, S. CRISPR-Cas13 mediated Knock Down in Drosophila cultured cells bioRxiv. doi: https://doi.org/10.1101/2020.11.01.364166

• Yu Z, Ren M, Wang Z, Zhang B, Rong YS, Jiao R, Gao G. Highly efficient genome modifications mediated by CRISPR/Cas9 in Drosophila. Genetics. 2013 Sep;195(1):289-91. doi: 10.1534/genetics.113.153825. Epub 2013 Jul 5. PMID: 23833182

• Zirin J, Hu Y, Liu L, Yang-Zhou D, Colbeth R, Yan D, Ewen-Campen B, Tao R, Vogt E, VanNest S, Cavers C, Villalta C, Comjean A, Sun J, Wang X, Jia Y, Zhu R, Peng P, Yu J, Shen D, Qiu Y, Ayisi L, Ragoowansi H, Fenton E, Efrem S, Parks A, Saito K, Kondo S, Perkins L, Mohr SE, Ni J, Perrimon N. Large-Scale Transgenic Drosophila Resource Collections for Loss- and Gain-of-Function Studies Genetics. 2020 Apr;214(4):755-767. doi: 10.1534/genetics.119.302964. Epub 2020 Feb 18. PMID: 32071193

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