Li Bin, Zhao Weiyu, Luo Xiao, Zhang Xinfu, Li Chenglong, Zeng Chunxi, Dong Yizhou
Division of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA.
Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville, FL 32610, USA.
Nat Biomed Eng. 2017 May;1(5). doi: 10.1038/s41551-017-0066. Epub 2017 May 10.
Cpf1, a type-V CRISPR-Cas effector endonuclease, exhibits gene-editing activity in human cells through a single RNA-guided approach. Here, we report the design and assessment of an array of 42 types of engineered Cpf1 (AsCpf1) CRISPR RNAs (crRNAs) and 5 types of AsCpf1 mRNAs, and show that the top-performing modified crRNA (cr3'5F, containing five 2'-fluoro ribose at the 3' termini) and AsCpf1 mRNA (full ψ-modification) improved gene-cutting efficiency by, respectively, 127% and 177%, with respect to unmodified crRNA and plasmid-encoding AsCpf1. We also show that the combination of cr3'5F and ψ-modified AsCpf1 or Cpf1 (LbCpf1) mRNAs augmented gene-cutting efficiency by over 300% with respect to the same control, and discovered that 11 out of 16 crRNAs from Cpf1 orthologs enabled genome editing in the presence of AsCpf1. Engineered CRISPR-Cpf1 systems should facilitate a broad range of genome editing applications.
Cpf1是一种V型CRISPR-Cas效应核酸内切酶，通过单一RNA引导方法在人类细胞中表现出基因编辑活性。在此，我们报告了42种工程化Cpf1（AsCpf1）CRISPR RNA（crRNA）和5种AsCpf1 mRNA的设计与评估，并表明表现最佳的修饰crRNA（cr3'5F，在3'末端含有五个2'-氟核糖）和AsCpf1 mRNA（完全ψ修饰）相对于未修饰的crRNA和编码AsCpf1的质粒，分别将基因切割效率提高了127%和177%。我们还表明，相对于相同对照，cr3'5F与ψ修饰的AsCpf1或Cpf1（LbCpf1）mRNA的组合将基因切割效率提高了300%以上，并且发现来自Cpf1直系同源物的16种crRNA中有11种在存在AsCpf1的情况下能够实现基因组编辑。工程化CRISPR-Cpf1系统应有助于广泛的基因组编辑应用。
