Programmable RNA N6-methyladenosine editing by CRISPR-Cas9 conjugates

Abstract

RNA modification in the form of N 6 -methyladenosine (m 6 A) regulates nearly all the post-transcriptional processes. The asymmetric m 6 A deposition suggests that regional methylation may have distinct functional consequences. However, current RNA biology tools do not distinguish the contribution of individual m 6 A modifications. Here we report the development of “m 6 A editing”, a powerful approach that enables m 6 A installation and erasure from cellular RNAs without changing the primary sequence. We engineered fusions of CRISPR-Cas9 and a single chain m 6 A methyltransferase that can be programmed with a guide RNA. The resultant m 6 A “writers” allow functional comparison of single site methylation in different mRNA regions. We further engineered m 6 A “erasers” by fusing CRISPR-Cas9 with ALKBH5 or FTO to achieve site-specific demethylation of RNAs. The development of programmable m 6 A editing not only expands the scope of RNA engineering, but also facilitates mechanistic understanding of epitranscriptome.

The emerging roles of N6-methyladenosine (m6A) deregulation in liver carcinogenesis

“…On the other hand, the RNA-targeting dCas9 can also be fused with the m6A demethylases FTO or ALKBH5 to erase site-specific m6A modification. It has been shown that removal of the m6A modification in lncRNA MALAT1 at A2577 resulted in structural change and altered the interaction with the RNA binding protein hnRNPC [87]. A similar dCas9-FTO system has also been reported by another group [88].…”

TRADES: Targeted RNA Demethylation by SunTag System

“…So we still lack a flexible approach to remove m 6 A with spatial and temporal specificity. Recently, some effective RNA m 6 A editing methods were reported by CRISPR-dCas9 conjugates and other programmable RNA binding proteins, [29][30][31] but with sufficient space for improvement.…”

“…[28] Recently, the fusion of CRISPR-dCas9 and m 6 A enzymes was developed for programmable m 6 A editing, which opens a new era to elucidate the precise role of RNA modification. [29] Rcas9 was also chosen as the RNA-targeting protein to realize sequence-specific m 6 A demethylation. [30] However, these technologies still need to be improved for two reasons.…”

Advances in biological functions of RNA chemical modification m<sup>6</sup>A

“…此外, VSV入侵还导致宿主降低 ALKBH5蛋白的活性, 进而影响免疫代谢产物衣康酸 的累积, 增强免疫反应, 抑制病毒复制 [105] . [107] . 使用RCas9和FTO的融合蛋白同样可以去除特 异位点上的m 6 A [108] .…”