RS-1 enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency

Abstract

Zinc-finger nuclease, transcription activator-like effector nuclease and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) are becoming major tools for genome editing. Importantly, knock-in in several non-rodent species has been finally achieved thanks to these customizable nucleases; yet the rates remain to be further improved. We hypothesize that inhibiting non-homologous end joining (NHEJ) or enhancing homology-directed repair (HDR) will improve the nuclease-mediated knock-in efficiency. Here we show that the in vitro application of an HDR enhancer, RS-1, increases the knock-in efficiency by two- to five-fold at different loci, whereas NHEJ inhibitor SCR7 has minimal effects. We then apply RS-1 for animal production and have achieved multifold improvement on the knock-in rates as well. Our work presents tools to nuclease-mediated knock-in animal production, and sheds light on improving gene-targeting efficiencies on pluripotent stem cells.

Figure 1. Effects of SCR7 and RS-1 on TALEN- or Cas9-mediated knock-in rates in vitro.

(a) Efficiency of Cas9-mediated RLL-EGFP knock-in in rabbit embryos. Left panel: effects of SCR7. Right panel: effects of RS-1 or RAD51 mRNA. (b) Effects of optimized RS-1 treatment (7.5 μM) on knock-in to CFTR and ApoAI loci in rabbit embryos versus control group (0 μM RS-1). Left panel: Cas9-mediated knock-in of CFTRdelF508 mutation to rabbit CFTR. Right panel: TALEN-mediated knock-in of hApoAII to rbApoAI. NS, not significantly different.

Figure 2. Effects of RS-1 on TALEN- or Cas9-mediated knock-in rates in vivo.

(a) Gene-targeting strategy of TALEN-mediated knock-in of hApoAII to rbApoAI locus. (b) Confirmation of hApoAII knock-in rabbits. Lanes 1–10: samples from individual kits. Upper: PCR products using primer set LF1/LR1. Lower: PCR products using primer set RF1/RR1. M, molecule weight marker. Arrows indicate knock-in band. +, positive for knock-in. −, negative for knock-in. Kits #1, 7 and 10 are identified as knock-in founders. (c) Three hApoAII knock-in founder rabbits (DOB: 12/5/2014). (d) Gene-targeting strategy of Cas9-mediated knock-in of EGFP to RLL locus. (e) Confirmation of RLL-EGFP knock-in rabbits. Lanes 1–13: samples from individual kits. Upper: PCR products using primer set LF2/LR2. Lower: PCR products using primer set RF2/RR2. M, molecule weight marker. Arrows indicate knock-in band. +, positive for knock-in. −, negative for knock-in. Kits #1, 2, 4, 9, 12 and 13 are identified as knock-in founders. (f) Three RLL-EGFP knock-in founder rabbits (DOB: 6/17/2014). (g) Comparison of frequency of WT, indel and knock-in alleles between embryos parented by founders of the RS-1 treatment group and the non-treated group. *P<0.05, χ²-test.