@article {1231110, title = {An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms}, journal = {Elife}, volume = {8}, year = {2019}, month = {2019 Nov 01}, abstract = {We previously reported a CRISPR-mediated knock-in strategy into introns of genes, generating an - transgenic library for multiple uses (Lee et al., 2018b). The method relied on double stranded DNA (dsDNA) homology donors with ~1 kb homology arms. Here, we describe three new simpler ways to edit genes in flies. We create single stranded DNA (ssDNA) donors using PCR and add 100 nt of homology on each side of an integration cassette, followed by enzymatic removal of one strand. Using this method, we generated GFP-tagged proteins that mark organelles in S2 cells. We then describe two dsDNA methods using cheap synthesized donors flanked by 100 nt homology arms and gRNA target sites cloned into a plasmid. Upon injection, donor DNA (1 to 5 kb) is released from the plasmid by Cas9. The cassette integrates efficiently and precisely . The approach is fast, cheap, and scalable.}, issn = {2050-084X}, doi = {10.7554/eLife.51539}, author = {Kanca, Oguz and Zirin, Jonathan and Garcia-Marques, Jorge and Knight, Shannon Marie and Yang-Zhou, Donghui and Amador, Gabriel and Chung, Hyunglok and Zuo, Zhongyuan and Ma, Liwen and He, Yuchun and Lin, Wen-Wen and Fang, Ying and Ge, Ming and Yamamoto, Shinya and Schulze, Karen L and Hu, Yanhui and Spradling, Allan C and Mohr, Stephanie E and Perrimon, Norbert and Bellen, Hugo J} }