Genome editing using CRISPR/Cas9 is changing the way we think about genome modification. The CRISPR system, because it relies on a small user-provide guide RNA (gRNA) to give the Cas9 endonuclease its sequence specificity, is easy to use, relative to, say, zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs).
The CRISPR system makes it possible to think about mutagenizing multiple genes in one experiment simultaneously, and this has been demonstrated in mice and rats. Liu et al (2014) demonstrate the feasibility of doing the same thing in Bombyx mori cells and embryos. While there was little doubt that multilocus targeting would be possible, it is nice to see it successfully accomplished – at least in cultured cells.
Liu et al. (2014) report a number of results that should provide useful information for others contemplating similar experiments. They show that the CRISPR system is very efficient at creating site-specific mutations in cultured B. mori cells. Germ-line CRISPR-based mutagenesis of B. mori was reported in 2013 by Wang et al so Liu et al.’s results in this case were not particularly novel.
They also demonstrate how CRISPRs can be used to create a 3kb deletion – something that has also been shown in Drosophila melanogaster.
Their most interesting result was their successful use of 10 gRNAs to simultaneously target Cas9 to 6 different genes.
Making, detecting and recovering lines of B. mori with multiple mutations was not reported but it seems doable.
Mutation detection without compromising your ability to start a line from insects with germ-line mutations can be particularly difficult. It appears that all of the enzymology will work just fine.
Highly efficient multiplex targeted mutagenesis and genomic structure variation in Bombyx mori cells using CRISPR/Cas9
Yuanyuan Liu1, Sanyuan Ma1, Xiaogang Wang, Jiasong Chang, Jie Gao, Run Shi, Jianduo Zhang, Wei Lu, Yue Liu, Ping Zhao, Qingyou Xia
Insect Biochemistry and Molecular Biology
Volume 49, June 2014, Pages 35–42