Methods

IRI-CCE: A Platform for Evaluating CRISPR-based Base Editing Tools and Its Components

2.1 E. coli strains

The four E. coli strains were used: 10-beta, DH5α, DB3.1, and BL21(DE3). The information about strain genotype provided in Supplementary Table 1. The primers and plasmids used in this study are listed in Supplementary Tables 2 and 3, respectively. The E. coli strains were cultured in Luria-Bertani (LB) broth with appropriate antibiotics.

2.2 Plasmid construction and cloning

The required bioparts were amplified by conventional polymerase chain reaction (PCR) using a Phusion high-fidelity DNA polymerase (Thermo Fisher Scientific, Waltham, MA). The cloning of different plasmid vectors was designed and performed by following the principle of MoClo (Weber et al., 2011) and Golden Gate assembly (Engler et al., 2014) protocols using BpiI/BsaI Type IIS enzyme digestion-ligation. The DNA recognition sites of restriction enzymes BsaI and BpiI were removed from internal sequences of bioparts during PCR amplification to make them suitable for Golden Gate cloning (a procedure also termed domestication). The DNA oligonucleotide pairs for the pEc1 (SJM901+RBSTL2) promoter and TerL3S2P21 terminator were annealed and ligated into BpiI-digested acceptor plasmids. Various promoters including pGlpT, p35S(Long), p35S(Short), pRbc, and pRPS5a were used from the pYTK001 (Addgene #65108) (Lee et al., 2015), pICH51266 (Addgene #50267) (Engler et al., 2014), pICH51277 (Addgene #50268) (Engler et al., 2014), pICH45195 (Addgene #50275) (Engler et al., 2014), and pKI1.1R (Addgene #85808) (Tsutsui and Higashiyama, 2017), respectively. The promoter DNA sequences and their evaluation provided in Supplementary Figures 1, 2, 3 and 4. Similarly, DNA sequences of Terminators, Ter35S, and TerHSP were adopted from the pICH41414 (Addgene #50337) (Engler et al., 2014) and pKI1.1R (Addgene #85808), respectively.

Three cytidine deaminases: PmCDA1 (Target-AID) (Nishida et al., 2016), evoCDA1 (Thuronyi et al., 2019), and APOBEC3A (Zong et al., 2018), were PCR amplified from PmCDA1-1x uracil-DNA glycosylase inhibitor protein (UGI) (Addgene #79620), evoCDA1 pBT277 (Addgene #122608), and A3A-PBE-ΔUGI (Addgene #119770), respectively. For all three deaminases, the same linker regions were used as reported in the previous studies. The ABE8e was synthesized from Bioneer Co. (Daejon, Korea), and the ABE9e variant (containing additional V82S/Q154R mutations) was cloned using ABE9e module as a template by site-directed mutagenesis PCR. PmCDA1-1xUGI was fused to the C terminus of nCas9. The evoCDA1, A3A, and ABE8e were fused to the N terminus of nCas9 with the XTEN linker. The 2xUGI module (template: Addgene #122608) was fused to the C terminus of evoCDA1-nCas9 and A3A-nCas9. The sgRNA expression was driven by bacterial pJ23119 (synthetically cloned) or plant AtU6 promoter (pICSL01009, Addgene #46968). The nCas9(D10A) was generated by the PCR method using previously optimized Cas9 as a template (Level 1 hCas9 module, Addgene #49771). Desired sgRNA sequence was PCR amplified using plasmid pICH86966::AtU6p::sgRNA_PDS (Addgene #46966) as a template and cloned together with either pJ23119 or pAtU6 for sgRNA expression.

For cloning of target region of the desired sgRNA sequence, a pair of oligonucleotide DNAs that contains the target sequence with PAM was annealed and ligated into BsmBI-digested universal target-acceptor plasmid L1 or L2 having optimized superfolder green fluorescent protein (sfGFP) (Lee et al., 2015) at downstream side (Supplementary Figure 5 and 6).

2.3 Bacterial transformation, plasmid isolation, and Sanger sequencing

Ligation products of all the steps during the cloning were transformed into competent cells of E. coli 10-beta strain by a heat-shock method. The bacterial culture was spread on the LB media containing desired antibiotic and incubated at 37°C for 18-24 h. The cloned plasmids and BE activities were confirmed by Sanger sequencing at Solgent Ltd. (Daejeon, Korea) or Cosmogentech Ltd. (Seoul, Korea). For mutagenesis assay, the different E. coli strains were transformed with the appropriate plasmids using the heat-shock method and were pre-cultured for 1 h with 1 ml of LB medium. After incubation for 1 h at 37°C, the fraction of cell cultures were spread on LB agar (1.5%) plates containing selection antibiotics with needed concentrations. The next day, individual colonies from the plate were inoculated in 3 ml LB broth with appropriate antibiotics and cultured at 37°C. The culture time was varied according to the experimental parameters and mentioned in appropriate sections. The plasmid isolation was done using Plasmid Mini-Prep Kit from BioFact Co. Ltd. (Daejeon, Korea) for Sanger sequencing analysis.

2.4 Promoter activity analysis

As shown in Figure 1B, all the tested promoters were cloned with sfGFP sequence at the downstream side, followed by a termination signal. Bacterial cultures were grown from the transformed single colonies in LB broth, shaking at 37°C for 24 h. The grown cultures were incubated for 24 h at 4°C, and then OD600 values were normalized to 1. The cells were harvested at 10000 rpm for 1 min and washed with 1x PBS (Phosphate buffered saline). Images were captured in blue light with a FluoroBox from CELLGENTEK Co., Ltd. (Deajeon, South Korea). The fluorescence intensity level was quantified using the ImageJ software (Schneider et al., 2012), and corrected total cell fluorescence (CTCF) was calculated using the following formula: CTCF = Integrated Density - (Area of selected cell x Mean fluorescence of background readings).

For qRT-PCR, RNA was extracted using the following method. E. coli-carrying plasmids (Figure 1B) were grown in LB medium. Cells were harvested during the exponential growth phase (12 h with OD600 value 0.5). Total RNA was extracted using RNeasy Protect Bacteria Mini Kit from Qiagen. For all samples, 600 ng of total RNA was used for complementary DNA (cDNA) synthesis using a QuantiTect Reverse Transcription Kit from Qiagen following the manufacturer’s instructions. To estimate the relative sfGFP transcript, the quantitative real-time PCR (qRT-PCR) reactions were carried out using the KAPA SYBR FAST qPCR kit from Kapa Biosystems (MA, USA) with sfGFP-specific primer sets (Supplementary Table 4). Cycling of PCR consisted of pre-denaturation at 95°C for 5 min followed by 40 cycles of a denaturation step at 95°C for 10 min, an annealing step at 60°C for 15 s, and final extension step at 72 °C for 20 s using the CFX384 Real-Time System from Bio-Rad, Hercules (CA, USA). The qRT-PCR reactions were performed with independent biological replicates. Relative sfGFP transcript values normalized against internal control 16S ribosomal RNA (rrsA) gene. Data analyses were performed by the 2−ΔΔCt method (Livak and Schmittgen, 2001).

2.5 Evaluation of editing activities

The single colonies were cultured, and the plasmid vectors containing synthetic targets were purified using Plasmid Mini-Prep Kit from BioFact Co. Ltd. (Daejeon, Korea) for further sequencing analysis. For sequencing analysis of genomic loci, the target fragments were PCR amplified using target-specific primers from the randomly picked colonies and then analyzed by Sanger sequencing. Sanger sequencing data analysis was performed using SnapGene software (GSL Biotech; available at snapgene.com). The editing efficiency was determined either by the ratio of non-edited to edited colonies from the randomly picked cells or by the nucleotide conversion rate using the online tool EditR (Kluesner et al., 2018) from C-to-T and A-to-G for CBE and ABE, respectively. The data were statistically analyzed and plotted in GraphPad Prism 9.0.0 (www.graphpad.com).

Article TitleIRI-CCE: A Platform for Evaluating CRISPR-based Base Editing Tools and Its Components

Abstract

Rapid assessment of CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated Cas protein)-based genome editing (GE) tools and their components are critical aspects for successful applications in different organisms. In many bacteria, double-stranded breaks (DSBs) generated by CRISPR/Cas tool generally cause cell death due to the lack of an efficient non-homologous end-joining pathway and restricts its use. CRISPR-based DSB-free base editors (BEs) have been applied for precise nucleotide editing in bacteria, which does not need to make DSBs. However, optimization of newer BE tools in bacteria is challenging owing to the toxic effects of BE reagents expressed using strong promoters. Improved variants of two main BEs capable of converting C-to-T (CBE) and A-to-G (ABE) have been recently developed but yet to be tested in bacteria. Here, we report a platform for in vivo rapid investigation of CRISPR-BE components in Escherichia coli (IRI-CCE) comprising different combinations of promoters/terminators. We demonstrate the use of IRI-CEE to characterize different variants of CBEs (PmCDA1, evoCDA1, APOBEC3A) and ABEs (ABE8e, ABE9e), exhibiting that each independent BE has its specific editing pattern for a given target site and promoter type. Additionally, the IRI-CCE platform offers a rapid way to screen functional gRNAs. In summary, CRISPR-BE components expressed by promoters of different strengths in the IRI-CCE allow an analysis of various BE tools, including cloned biopart modules and gRNAs.


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