Engineering CRISPR/Cpf1 with tRNA promotes genome editing capability in mammalian systems
- 1.1k Downloads
CRISPR/Cpf1 features a number of properties that are distinct from CRISPR/Cas9 and provides an excellent alternative to Cas9 for genome editing. To date, genome engineering by CRISPR/Cpf1 has been reported only in human cells and mouse embryos of mammalian systems and its efficiency is ultimately lower than that of Cas9 proteins from Streptococcus pyogenes. The application of CRISPR/Cpf1 for targeted mutagenesis in other animal models has not been successfully verified. In this study, we designed and optimized a guide RNA (gRNA) transcription system by inserting a transfer RNA precursor (pre-tRNA) sequence downstream of the gRNA for Cpf1, protecting gRNA from immediate digestion by 3′-to-5′ exonucleases. Using this new gRNAtRNA system, genome editing, including indels, large fragment deletion and precise point mutation, was induced in mammalian systems, showing significantly higher efficiency than the original Cpf1-gRNA system. With this system, gene-modified rabbits and pigs were generated by embryo injection or somatic cell nuclear transfer (SCNT) with an efficiency comparable to that of the Cas9 gRNA system. These results demonstrated that this refined gRNAtRNA system can boost the targeting capability of CRISPR/Cpf1 toolkits.
KeywordsCRISPR/Cpf1 gRNAtRNA system Genome editing Rabbit Pig
This work was supported by Grants from the National Natural Science Foundation of China (81702115, 81672317), National Key R&D Program of China (2017YFA0105103, 2017YFA0105101), Bureau of International Cooperation, The Chinese Academy of Sciences (154144KYSB20150033), the Science and Technology Planning Project of Guangdong Province, China (2014B020225003, 2016A030303046, 2015B020229002, 2016A030313169, 2016B030229008), the Youth Innovation Promotion Association, CAS (2017409), Pearl River S&T Nova Program of Guangzhou (201710010112), the Bureau of Science and Technology of Guangzhou Municipality (201704030034), the Science and Technology Planning Project of Guangdong Province, China (2017B030314056).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
Supplementary material 9 (MP4 26852 kb)
- 23.Orlando SJ, Santiago Y, DeKelver RC, Freyvert Y, Boydston EA, Moehle EA, Choi VM, Gopalan SM, Lou JF, Li J (2010) Zinc-finger nuclease-driven targeted integration into mammalian genomes using donors with limited chromosomal homology. Nucleic Acids Res 38(15):e152–e152CrossRefPubMedPubMedCentralGoogle Scholar
- 27.Xin JG, Yang HQ, Fan NN, Zhao BT, Ouyang Z, Liu ZM, Zhao Y, Li XP, Song J, Yang Y et al (2013) Highly efficient generation of GGTA1 biallelic knockout inbred mini-pigs with TALENs. PLoS One 8(12):9Google Scholar
- 33.Haghighi K, Kolokathis F, Gramolini AO, Waggoner JR, Pater L, Lynch RA, Fan GC, Tsiapras D, Parekh RR, Dorn GW et al (2006) A mutation in the human phospholamban gene, deleting arginine 14, results in lethal, hereditary cardiomyopathy. Proc Natl Acad Sci USA 103(5):1388–1393CrossRefPubMedGoogle Scholar
- 42.Singh D, Mallon J, Poddar A, Wang Y, Tipanna R, Yang O, Bailey S, Ha T (2017) Real-time observation of DNA target interrogation and product release by the RNA-guided endonuclease CRISPR Cpf1. https://doi.org/10.1101/205575