Points of View on the Tools for Genome/Gene Editing

Abstract

Theoretically, a DNA sequence-specific recognition protein that can distinguish a DNA sequence equal to or more than 16 bp could be unique to mammalian genomes. Long-sequence-specific nucleases, such as naturally occurring Homing Endonucleases and artificially engineered ZFN, TALEN, and Cas9-sgRNA, have been developed and widely applied in genome editing. In contrast to other counterparts, which recognize DNA target sites by the protein moieties themselves, Cas9 uses a single-guide RNA (sgRNA) as a template for DNA target recognition. Due to the simplicity in designing and synthesizing a sgRNA for a target site, Cas9-sgRNA has become the most current tool for genome editing. Moreover, the RNA-guided DNA recognition activity of Cas9-sgRNA is independent of both of the nuclease activities of it on the complementary strand by the HNH domain and the non-complementary strand by the RuvC domain, and HNH nuclease activity null mutant (H₈₄₀A) and RuvC nuclease activity null mutant (D₁₀A) were identified. In accompaniment with the sgRNA, Cas9, Cas9(D₁₀A), Cas9(H₈₄₀A), and Cas9(D₁₀A, H₈₄₀A) can be used to achieve double strand breakage, complementary strand breakage, non-complementary strand breakage, and no breakage on-target site, respectively. Based on such unique characteristics, many engineered enzyme activities, such as DNA methylation, histone methylation, histone acetylation, cytidine deamination, adenine deamination, and primer-directed mutation, could be introduced within or around the target site. In order to prevent off-targeting by the lasting expression of Cas9 derivatives, a lot of transient expression methods, including the direct delivery of Cas9-sgRNA riboprotein, were developed. The issue of biosafety is indispensable in in vivo applications; Cas9-sgRNA packaged into virus-like particles or extracellular vesicles have been designed and some in vivo therapeutic trials have been reported.

Keywords: CRISPR; Cas9; extracellular vesicles; genome/gene-editing.

Figure 1

Diagrams of SpCas9 and its derivatives for various applications. The domain organization of SpCas9 (A) and a schematic diagram of wild-type SpCas9 associated with a sgRNA (B) was illustrated. The non-complementary strand is cut by the RuvC nuclease domain, and this nuclease activity was blocked in D₁₀A mutant. On the other hand, the complementary strand was digested by the HNH nuclease domain, and such nuclease activity was destroyed in H₈₄₀A mutant. (C) The D₁₀A mutant, also named Cas9 nickase (nCas9), was engineered as a C to T nucleotide editor by linking a cytidine deaminase, APOBEC1, on the N-terminus of it and the switching probability could be elevated by the fusion of a uracil glycosylase inhibitor (UGI) on the C-terminus of nCas9. Like TALE, dCas9 could be guided by a sgRNA as a sequence-specific DNA-binding riboprotein. Transcriptional regulators, DNA modification enzymes, or histone modification enzymes could be fused to either or both of the N- and C-termini. In case of reverse transcriptase, it was fused to the C-terminus of Cas9, accompanied by an RNA template with 3′-end complementary to the non-complementary strand of protospacer, which could alter the nearby nucleotides downstream the RuvC cutting site (D). The localization of the Cas9-sgRNA also could be guided to an X-protein through an FKBP12–rapamycin–FRB bridge (E). The localization of the Cas9-sgRNA could also be guided via certain specific interactions, such as those between aptamer RNA and ABP. The tetraloop was replaced by an RNA aptamer of unique secondary structure, which can be recognized by a specific aptamer binding protein (F).