Performance of the Cas9 Nickase System in Drosophila melanogaster

Recent studies of the Cas9/sgRNA system in Drosophila melanogaster genome editing have opened new opportunities to generate site-specific mutant collections in a high-throughput manner. However, off-target effects of the system are still a major concern when analyzing mutant phenotypes. Mutations converting Cas9 to a DNA nickase have great potential for reducing off-target effects in vitro. Here, we demonstrated that injection of two plasmids encoding neighboring offset sgRNAs into transgenic Cas9D10A nickase flies efficiently produces heritable indel mutants. We then determined the effective distance between the two sgRNA targets and their orientations that affected the ability of the sgRNA pairs to generate mutations when expressed in the transgenic nickase flies. Interestingly, Cas9 nickase greatly reduces the ability to generate mutants with one sgRNA, suggesting that the application of Cas9 nickase and sgRNA pairs can almost avoid off-target effects when generating indel mutants. Finally, a defined piwi mutant allele is generated with this system through homology-directed repair. However, Cas9D10A is not as effective as Cas9 in replacing the entire coding sequence of piwi with two sgRNAs.

[1]  Simon L. Bullock,et al.  Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila , 2014, Proceedings of the National Academy of Sciences.

[2]  J. Haug,et al.  Piwi Is Required in Multiple Cell Types to Control Germline Stem Cell Lineage Development in the Drosophila Ovary , 2014, PloS one.

[3]  R. Jiao,et al.  Various applications of TALEN- and CRISPR/Cas9-mediated homologous recombination to modify the Drosophila genome , 2014, Biology Open.

[4]  Y. Rong,et al.  Efficient Gene Knock-out and Knock-in with Transgenic Cas9 in Drosophila , 2014, G3: Genes, Genomes, Genetics.

[5]  Jennifer A. Doudna,et al.  Structures of Cas9 Endonucleases Reveal RNA-Mediated Conformational Activation , 2014, Science.

[6]  F. Storici,et al.  To Nick or Not to Nick: Comparison of I-SceI Single- and Double-Strand Break-Induced Recombination in Yeast and Human Cells , 2014, PloS one.

[7]  Feng Zhang,et al.  Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA , 2014, Cell.

[8]  C. Rubinstein,et al.  Highly Specific and Efficient CRISPR/Cas9-Catalyzed Homology-Directed Repair in Drosophila , 2014, Genetics.

[9]  Ying Peng,et al.  A simplified and efficient germline-specific CRISPR/Cas9 system for Drosophila genomic engineering , 2013, Fly.

[10]  Jin-Soo Kim,et al.  Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases , 2014, Genome research.

[11]  C. Alexandre,et al.  Accelerated homologous recombination and subsequent genome modification in Drosophila , 2013, Development.

[12]  Jianzhong Xi,et al.  Optimized gene editing technology for Drosophila melanogaster using germ line-specific Cas9 , 2013, Proceedings of the National Academy of Sciences.

[13]  Shu Kondo,et al.  Highly Improved Gene Targeting by Germline-Specific Cas9 Expression in Drosophila , 2013, Genetics.

[14]  Bo Zhang,et al.  Highly Efficient Genome Modifications Mediated by CRISPR/Cas9 in Drosophila , 2013, Genetics.

[15]  Eli J. Fine,et al.  DNA targeting specificity of RNA-guided Cas9 nucleases , 2013, Nature Biotechnology.

[16]  David A. Scott,et al.  Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity , 2013, Cell.

[17]  E. Lai,et al.  Drosophila piwi Mutants Exhibit Germline Stem Cell Tumors that Are Sustained by Elevated Dpp Signaling , 2013, Current Biology.

[18]  G. Church,et al.  CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering , 2013, Nature Biotechnology.

[19]  Melissa M. Harrison,et al.  Genome Engineering of Drosophila with the CRISPR RNA-Guided Cas9 Nuclease , 2013, Genetics.

[20]  Chris P. Ponting,et al.  Highly Efficient Targeted Mutagenesis of Drosophila with the CRISPR/Cas9 System , 2013, Cell reports.

[21]  J. Keith Joung,et al.  High frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells , 2013, Nature Biotechnology.

[22]  James E. DiCarlo,et al.  RNA-Guided Human Genome Engineering via Cas9 , 2013, Science.

[23]  A. Pingoud,et al.  Site- and strand-specific nicking of DNA by fusion proteins derived from MutH and I-SceI or TALE repeats , 2013, Nucleic acids research.

[24]  Le Cong,et al.  Multiplex Genome Engineering Using CRISPR/Cas Systems , 2013, Science.

[25]  Shuang-yong Xu,et al.  Natural zinc ribbon HNH endonucleases and engineered zinc finger nicking endonuclease , 2012, Nucleic acids research.

[26]  R. Barrangou,et al.  Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria , 2012, Proceedings of the National Academy of Sciences.

[27]  J. Doudna,et al.  A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity , 2012, Science.

[28]  Eunji Kim,et al.  Precision genome engineering with programmable DNA-nicking enzymes , 2012, Genome research.

[29]  Claudio Mussolino,et al.  Engineered zinc finger nickases induce homology-directed repair with reduced mutagenic effects , 2012, Nucleic acids research.

[30]  J. Doudna,et al.  RNA-guided genetic silencing systems in bacteria and archaea , 2012, Nature.

[31]  R. Barrangou,et al.  CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. , 2011, Annual review of genetics.

[32]  N. Maizels,et al.  DNA Nicks Promote Efficient and Safe Targeted Gene Correction , 2011, PloS one.

[33]  David J. Rawlings,et al.  Tracking genome engineering outcome at individual DNA breakpoints , 2011, Nature Methods.

[34]  R. Terns,et al.  CRISPR-based adaptive immune systems. , 2011, Current opinion in microbiology.

[35]  J. Vogel,et al.  CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III , 2011, Nature.

[36]  B. Stoddard,et al.  Single-strand nicks induce homologous recombination with less toxicity than double-strand breaks using an AAV vector template , 2010, Nucleic acids research.

[37]  Barry L. Stoddard,et al.  Natural and engineered nicking endonucleases—from cleavage mechanism to engineering of strand-specificity , 2010, Nucleic Acids Res..

[38]  H. Deveau,et al.  CRISPR/Cas system and its role in phage-bacteria interactions. , 2010, Annual review of microbiology.

[39]  A. D. de Vries,et al.  Stimulation of homology-directed gene targeting at an endogenous human locus by a nicking endonuclease , 2009, Nucleic acids research.

[40]  Julius Brennecke,et al.  Specialized piRNA Pathways Act in Germline and Somatic Tissues of the Drosophila Ovary , 2009, Cell.

[41]  Ryo Takeuchi,et al.  Generation of a nicking enzyme that stimulates site-specific gene conversion from the I-AniI LAGLIDADG homing endonuclease , 2009, Proceedings of the National Academy of Sciences.

[42]  Broome,et al.  Literature cited , 1924, A Guide to the Carnivores of Central America.

[43]  N. Perrimon,et al.  Vector and parameters for targeted transgenic RNA interference in Drosophila melanogaster , 2008, Nature Methods.

[44]  Haifan Lin,et al.  The Role of PIWI and the miRNA Machinery in Drosophila Germline Determination , 2006, Current Biology.

[45]  N. Lau,et al.  Characterization of the piRNA Complex from Rat Testes , 2006, Science.

[46]  C. Sander,et al.  A novel class of small RNAs bind to MILI protein in mouse testes , 2006, Nature.

[47]  Ravi Sachidanandam,et al.  A germline-specific class of small RNAs binds mammalian Piwi proteins , 2006, Nature.

[48]  Haifan Lin,et al.  A novel class of small RNAs in mouse spermatogenic cells. , 2006, Genes & development.

[49]  Haifan Lin,et al.  Regulatory Relationship among piwi, pumilio, and bag-of-marbles in Drosophila Germline Stem Cell Self-Renewal and Differentiation , 2005, Current Biology.

[50]  E. Wimmer,et al.  Highly sensitive, fluorescent transformation marker for Drosophila transgenesis , 2000, Development Genes and Evolution.

[51]  Haifan Lin,et al.  A novel class of evolutionarily conserved genes defined by piwi are essential for stem cell self-renewal. , 1998, Genes & development.

[52]  A. Spradling,et al.  A novel group of pumilio mutations affects the asymmetric division of germline stem cells in the Drosophila ovary. , 1997, Development.