Heritable gene targeting in the mouse and rat using a CRISPR-Cas system

Generating rats with conditional alleles using CRISPR/Cas9

“…We also examined Dnmt1-A OTS-3 and Dnmt3b-B OTS-9 in the other corresponding founders by the T7EN1 cleavage assay and sequencing, and found that mutations at these 2 sites indeed occurred in 7 Dnmt1 (7/12) and 9 Dnmt3b (9/30) founders, respectively (Supplementary information, Figure S9). Recent reports have also indicated that CRISPR/Cas9 induced off-target effects at a very low level in mouse and rat, suggesting that the potential off-target effect may not be a major concern for the application of the CRISPR/Cas9 system in genome modification [4][5]7]. In summary, we described here for the first time the generation of rats carrying conditional alleles using the CRISPR/Cas9 system combined with a single circular donor vector.…”

“…Several genome-editing technologies, such as zinc-finger nucleases (ZFNs) [1][2], transcription activator-like effector nucleases (TALENs) [3] and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated 9 (Cas9) system [4][5], have been used to produce knockout rat models by generating DNA double-strand breaks (DSBs) followed by non-homologous end joining (NHEJ)-mediated repair. However, the simple knockout strategies have limits for studying genes that are critical for embryogenesis.…”

“…A recent report demonstrated that rats carrying a floxed gene can be successfully produced via microinjection of 2 pairs of ZFNs and 2 plasmid donors into fertilized eggs [2]. In comparison with ZFNs or TALENs, CRISPR/ Cas9 provides a simpler way to edit the eukaryotic genome even in a multiplex manner [4][5]. Recently, mice carrying conditional alleles have been generated by using CRISPR/Cas9 system [7].…”

“…Recent reports have suggested that the CRISPR/Cas9 system may tolerate sequence mismatches, and thereby generate off-target mutations [4][5]7]. Therefore, we comprehensively investigated the potential off-target effects in mutant founders.…”

Human Germline Genome Editing

“…It has been shown that engineered nucleases, especially CRISPR/Cas9, can be easily used to edit genes in mammalian embryos such as mice, rats, and even monkeys. 11,13,14 These embryos can then be implanted into foster animals and carried to term, generating live-born animals carrying precise changes in their DNA. However, off-target mutagenesis and mosaicism in the resulting animals can be significant drawbacks of the technology.…”