Methods

Highly specific chimeric DNA-RNA guided genome editing with enhanced CRISPR-Cas12a system
CRISPR/Cas12a Molecular Biology

Preparation of the CRISPR-Cas12a recombinant protein and chimeric guides

wt- and en-AsCas12a recombinant proteins were prepared for the in vitro DNA cleavage assay. Codon-optimized AsCas12a (Acidaminococcus sp. Cas12a) coding sequence was cloned into a pET28a bacterial expression vector and then transformed into BL21 (DE3) Escherichia coli cells. Transformed bacterial colonies were cultured at 37 °C until the optical density reached 0.6, after which isopropylthio-β-galactoside (IPTG) inoculation was performed. After 48 h, E. coli cells were precipitated at 4 °C and 5,000 rpm, following which the culture medium of the upper layer was removed. The precipitated E. coli cell pellet was resuspended in lysis buffer 10 mM β-mercaptoethanol, 300 mM NaCl, 20 mM Tris-HCl (pH8.0), 1 mM PMSF, and 1% TritonX-100. In order to disturb the bacterial cell membrane, sonication was performed on ice water for 3 min, following which the cell lysate was precipitated for 10 min at 5,000 rpm at 4 °C. Next, the nitrilotriacetic acid (Ni-NTA) resin was pre-washed with wash buffer 20mM Tris-HCl (pH 8.0), 300 nM NaCl and the precipitated cell lysate was stirred at 4 °C for 90 min. Washing was performed with ten times the volume of wash buffer to remove non-specific binding components in the mixed cell lysate solution. For the elution of AsCas12a protein, an elution buffer 20 mM Tris-HCl (pH 8.0), 300 nM NaCl, 200 mM imidazole was used and finally exchanged against the storage buffer 200 mM NaCl, 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; pH 7.5), 1 mM dithiothreitol (DTT), 40% glycerol using a Centricon (Millipore, Amicon® Ultra-15), and stored at -80 °C. Chimeric DNA-RNA oligonucleotides (Bioneer) were synthesized for each target gene sequence and dissolved in diethyl pyrocarbonate (DEPC) water and then stored at -80 °C (Supplementary Table S1).

Preparation of the guide RNA for Cas12a and nCas9(D10A)

Guide RNAs for Cas12a and nCas9(D10A) were generated by in vitro transcription. A DNA template for in vitro transcription was constructed using annealing or extension polymerase chain reaction (PCR) with sense and antisense DNA oligonucleotides (Macrogen) containing the target DNA sequence (Supplementary Table S2). DNA templates were mixed with T7 RNA polymerase (NEB, M0251L) and reaction buffer mixture (50 mM MgCl2, 100 mM ribonucleoside tri-phosphate (rNTP; Jena Bio, NU-1014), 10X RNA polymerase reaction buffer, 100 mM DTT, RNase inhibitor Murine, DEPC), and incubated at 37 °C. After 16 h, to remove the original DNA template, DNase I was added and the mixture was incubated for another 1 h at 37°C. The transcribed RNA was purified using a column (MP Biomedicals, GENECLEAN® Turbo Kit). The purified RNA was concentrated through lyophilization (2,000 rpm) at -55 °C for 1 h.

In vitro DNA cleavage assay

On-/off-target site PCR amplicons of each gene (DNMT1, CCR5, IL2A-AS1, and AAVS1) were obtained from purified human genomic DNA using DNA primers (Supplementary Table S2). The purified target PCR amplicon was incubated with purified recombinant wt- or en-Cas12a protein and crRNA (RNA-guides or chimeric DNA-RNA guides) in 10X buffer (NEBuffer3.1, NEB) for 1 h. After adding a stop buffer 100 mM ethylenediaminetetraacetic acid (EDTA), 1.2% sodium dodecyl sulfate (SDS) to stop the reaction, the cleaved fragment was separated using 2% agarose gel electrophoresis. DNA cleavage efficiency (cleaved fragment intensity %/total fragment intensity %) was measured using ImageJ software (NIH).

Cell culture and transfection

The HEK293FT (ATCC) cell line was cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco) with 10% fetal bovine serum (FBS, Gibco) at 37 °C and in 5% CO2. To ensure efficient chimeric DNA-RNA guide delivery, we performed sequential transfection with wt- or en-Cas12a expression vectors and crRNAs. For the primary transfection, 105 cells were mixed with a plasmid vector (AsCas12a, n-SpCas9 (D10A, H840A)) and 20 μl of electroporation buffer (Lonza, V4XC-2032) and were nucleofected according to the manufacturer’s instructions (program: CM-137). The transfected cells were transferred to a 24-well plate with 500 μl of media and incubated at 37 °C in 5% CO2. Twenty four hours after primary transfection, for secondary transfection, crRNA (200 pmol), single guide RNA (sgRNA; 30 pmol), 1 μl P3000, and 1.5 μl Lipofectamine 3000 reagent (Thermo) were mixed in 50 μl Opti-MEM (Gibco), incubated for 10 min, and added to DMEM media. Forty-eight hours after the second transfection, cells were harvested and genomic DNA was extracted using a genomic DNA purification kit (Qiagen, DNeasy Blood & Tissue Kit).

Deep sequencing and data analysis

To analyze the indel frequency of the on-/off-target locus of each gene, targeted deep sequencing was performed using PCR amplicons. The Cas-OFFinder (http://www.rgenome.net/cas-offinder/) web tool was used to select potential off-target sites corresponding to each on-target site. For the preparation of PCR amplicons, PCR amplification was performed using DNA primers corresponding to each endogenous locus (Supplementary Table S2). To add adapter and index sequences to each 5’ and 3’ ends, nested PCR was performed using Phusion™ High-Fidelity DNA Polymerase (Thermo). After index tagging, the PCR amplicon mixture was analyzed using a Mini-Seq (Illumina, SY-420-1001) according to the manufacturer’s guidelines. Sequencing read fatstq files were analyzed using Cas-Analyzer (http://www.rgenome.net/cas-analyzer/), and the indel ratio (mutant DNA read number/total DNA read number) was calculated.

Article TitleHighly specific chimeric DNA-RNA guided genome editing with enhanced CRISPR-Cas12a system

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a system is composed of a Cas12a effector that acts as a deoxyribonucleic acid (DNA)-cleaving endonuclease and a crispr ribonucleic acid (crRNA) that guides the effector to the target DNA. It is considered a key molecule for inducing target-specific gene editing in various living systems. Here, we improved the efficiency and specificity of the CRISPR-Cas12a system through protein and crRNA engineering. In particular, to optimize the CRISPR-Cas12a system at the molecular level, we used a chimeric DNA-RNA guide chemically similar to crRNA to maximize target sequence specificity. Compared to the wild type (wt)-Cas12a system, when using enhanced Cas12a system (en-Cas12a), the efficiency and target specificity improved on average by 7.41 and 7.60 times respectively. In our study, when the chimeric DNA-RNA guided en-Cas12a effector was used, the gene editing efficiency and accuracy were simultaneously increased. These findings could contribute to highly accurate genome editing, such as human gene therapy, in the near future.


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