Chemical Control of CRISPR Gene Editing via Conditional Diacylation Crosslinking of Guide RNAs

Abstract Conditional control of RNA structure and function has emerged as an effective toolkit. Here, a strategy based on a one‐step introduction of diacylation linkers and azide groups on the 2′‐OH of RNA is advance. Selected from eight phosphine reagents, it is found that 2‐(diphenylphosphino)ethylamine has excellent performance in reducing azides via a Staudinger reduction to obtain the original RNA. It is demonstrated that the enzymatic activities of Cas13 and Cas9 can be regulated by chemically modified guide RNAs, and further achieved ligand‐induced gene editing in living cells by a controllable CRISPR/Cas9 system.


Synthesis of HBC525 and HBC620
The synthesis of HBC525 and HBC620 were conducted according to previous literatures. [2] Table of contents   Chemical synthesis  Page S2-6   Table of contents  Page S7-9 Experimental section Page S9 Table S1 DNA and RNA sequences used in the current study Page S10-11

Figure S1
Schematic illustration of target SLX4IP DNA sequence Page S12

Figure S3
Schematic illustration of target HBEGF DNA sequence, target HPRT1 DNA sequence, target t-GFP1 DNA sequence and target t-GFP2 DNA sequence Page S13-14

Figure S11
Crosslinking strategy for regulating RNA-RNA interaction Page S19

Figure S12
Crosslinking strategy for controlling RNA folding Page S20

Figure S13
Crosslinking strategy for regulating RNA hybridization Page S20

Experimental section
Crosslinking strategy for controlling Pepper aptamer folding. In this assay, RNA aptamer named Pepper was used to test the crosslinked Pepper disturbing the recognition between aptamer and fluorescence molecules (HBC620 and HBC525). Crosslinked Pepper was separately incubated with HBC525 or HBC620 (10 μM) in folding buffer (5 mM MgCl2, 100 mM KCl, 40 mM HEPES, pH 7.4 @ 25℃) at 37℃ for 2 h. Pepper without any treatment was used as a control in this assay. Then the fluorescence intensity was analyzed on LS-55 FL Spectrophotometer (Perkin Elmer). For fluorescence recovery assay mediated by DPPEA, the crosslinked Pepper was firstly incubated with DPPEA of different concentrations in RNase-free water at 37℃ for 4 h, and then the samples were incubated with HBC and analyzed as before. Crosslinking strategy for controlling enzyme recognition. Bst DNA pol assay: Untreated R-32 nt (34 ng) or crosslinked R-32 nt (12.5 mM BIN2 for various of time), dNTPs, DNA primer (11 ng) 5' end with FAM labeling and Bst DNA pol (2.4 U) were incubated at 42℃ for 3 h in reaction buffer (10 mM KCl,10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 20 mM Tris-HCl, pH 8.8 @ 25℃). M-MuLV RT: Untreated R-32 nt (34 ng) or crosslinked R-32 nt (12.5 mM BIN2 for various of time), dNTPs, DNA primer (11 ng) 5' end with FAM labeling and M-MuLV RT (2.4 U) were incubated at 37℃ for 3 h in reaction buffer (3 mM MgCl2, 75 mM KCl, 50 mM Tris-HCl, pH 8.3 @ 25℃).
The recovery assays of the reverse transcription were carried out by treating crosslinked R-32 nt with different concentrations of DPPEA at 37℃ for 4 h, and the following procedures are similar to before. Cas13a, dCas9 and dCas13a of expression and purification. This part of the experiment procedure is similar to the recently published studies. [3] Supplementary Table 1

Name
Sequence (from 5' to 3') Construct The forward primer for sg-SLX4IP  Figure S1. Schematic illustration of target SLX4IP DNA sequence. The target site and PCR primer were indicated by green color and underlining, respectively. Red asterisk displayed the cleavage sites by Cas9 nuclease. Schematic illustration of the sequence of SLX4IP gene around target loci. The SLX4IP gene is located on the short arm (p) of chromosome 20 at position 12. 2 (20p12.2). We generated target SLX4IP DNA (t-SLX4IP) carrying the target loci from Hela-OC genomic DNA. The 20 nt sequence was the same sequence as the target sequence.