Abstract
Cpf1 is an RNA-guided endonuclease that can be programmed to cleave DNA targets. Specific features, such as containing a short crRNA, creating a staggered cleavage pattern and having a low off-target rate, render Cpf1 a promising gene-editing tool. Here, we present a new Cpf1 ortholog, EeCpf1, as a genome-editing tool; this ortholog is derived from the gut bacterial species Eubacterium eligens. EeCpf1 exhibits a higher cleavage activity with the Mn2+ metal cofactor and efficiently cuts the target DNA with an engineered, nucleotide extended crRNA at the 5′ target site. When mouse blastocysts were injected with multitargeting crRNAs against the IL2R-γ gene, an essential gene for immunodeficient mouse model production, EeCpf1 efficiently generated IL2R-γ knockout mice. For the first time, these results demonstrate that EeCpf1 can be used as an in vivo gene-editing tool for the production of knockout mice. The utilization of engineered crRNA with multiple target sites will help to explore the in vivo DNA cleavage activities of Cpf1 orthologs from other species that have not been demonstrated.
Highlights
Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems eliminate invading genetic elements in prokaryotes[1]
The conservation of the stem-loop scaffolds indicates that the EeCpf[1] protein may recognize the 5′ T-rich protospacer adjacent motif (PAM) sequence according to previous data[11]
We presented the in vivo DNA cleavage activity of Cpf[1] from E. eligens for the first time; we used this activity for gene editing to produce knockout mice
Summary
Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems eliminate invading genetic elements in prokaryotes[1]. CRISPR/Cas effector proteins are RNA-guided endonucleases that can be programmed to cleave DNA or/and RNA targets. Cas[9] makes a blunt cut adjacent to the PAM, while Cpf[1] generates a 5 base pair (bp) staggered cut 17 nt downstream of the PAM9 These specific features of Cpf[1], together with the feature of lower off-target cleavage rates compared to those of Cas[9], can broaden the spectrum of genome editing to various fields[10]. NC3005 (BsCpf1)) were shown to have DNA cleavage functions in vivo, while the Cpf[1] variants of FnCpf[1], AsCpf[1] and LbCpf[1] have been studied most intensively and have been used as gene-editing tools[13,14]. We show the in vivo DNA cleavage activity of EeCpf[1] for the first time and present it as an efficient genome-editing tool
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