Evaluation of Methods to Assess in vivo Activity of Engineered Genome-Editing Nucleases in Protoplasts

Abstract

Genome-editing is being implemented in increasing number of plant species using engineered sequence specific nucleases (SSNs) such as Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated systems (CRISPR/Cas9), Transcription activator like effector nucleases (TALENs), and more recently CRISPR/Cas12a. As the tissue culture and regeneration procedures to generate gene-edited events are time consuming, large-scale screening methodologies that rapidly facilitate validation of genome-editing reagents are critical. Plant protoplast cells provide a rapid platform to validate genome-editing reagents. Protoplast transfection with plasmids expressing genome-editing reagents represents an efficient and cost-effective method to screen for in vivo activity of genome-editing constructs and resulting targeted mutagenesis. In this study, we compared three existing methods for detection of editing activity, the T7 endonuclease I assay (T7EI), PCR/restriction enzyme (PCR/RE) digestion, and amplicon-sequencing, with an alternative method which involves tagging a double-stranded oligodeoxynucleotide (dsODN) into the SSN-induced double stranded break and detection of on-target activity of gene-editing reagents by PCR and agarose gel electrophoresis. To validate these methods, multiple reagents including TALENs, CRISPR/Cas9 and Cas9 variants, eCas9(1.1) (enhanced specificity) and Cas9-HF1 (high-fidelity1) were engineered for targeted mutagenesis of Acetolactate synthase1 (ALS1), 5-Enolpyruvylshikimate- 3-phosphate synthase1 (EPSPS1) and their paralogs in potato. While all methods detected editing activity, the PCR detection of dsODN integration provided the most straightforward and easiest method to assess on-target activity of the SSN as well as a method for initial qualitative evaluation of the functionality of genome-editing constructs. Quantitative data on mutagenesis frequencies obtained by amplicon-sequencing of ALS1 revealed that the mutagenesis frequency of CRISPR/Cas9 reagents is better than TALENs. Context-based choice of method for evaluation of gene-editing reagents in protoplast systems, along with advantages and limitations associated with each method, are discussed.

Keywords: CRISPR/Cas9; NHEJ; TALENs; double-stranded oligodeoxynucleotides; genome-editing; protoplasts.

FIGURE 1

Constructs for targeted mutagenesis of ALS1 and EPSPS1 loci in potato protoplasts using CRISPR/Cas9 and TALEN reagents. (A) Map of potato target gene Acetolactate synthase1 (ALS1) and (B) 3-Phosphoshikimate 1-carboxyvinyltransferase (EPSPS1) used in the study. The sequence of the target site is shown with sgRNA spacer in red, PAM in blue, TALEN binding sites are underlined, and restriction enzyme sites are in bold italicized. (C) Structure of the constructs used for expressing sgRNAs and TALENs co-expressing green fluorescent protein (GFP) for both target genes. PCaMV35S, cauliflower mosaic virus 35S promoter, AtCas9, Arabidopsis codon optimized Cas9 nuclease; tHSP, Arabidopsis heat shock protein 18.2 terminator; AtU6, Arabidopsis U6 promoter; sgRNA, single guide RNA; PFMV34S, figwort mosaic virus 34S promoter; GFP, green fluorescent protein; tE9, pea ribulose bisphosphate carboxylase small subunit terminator; P2A, ribosomal skipping sequence.

FIGURE 2

Determination of protoplast viability and transformation efficiency of genome-editing nucleases targeting ALS1. (a) Protoplasts isolated from in vitro grown potato leaves. (b) Protoplasts stained with Evans blue to test viability. Arrows indicate defective protoplast cells into which the dye permeated. Scale bar = 20 μ. (c) Transformation efficiencies are compared between the CRISPR/Cas9 and TALEN plasmid constructs targeting ALS1. Each bar represents the % mean value of three independent transformations, each with five technical replicates ± Standard deviation, Student’s t-tests (P ≤ 0.05). (d–f) Confocal laser scanning microscope images showing, merged images of GFP fluorescence (green) and bright field (gray). CRISPR/Cas9 targeting ALS1 (d), TALENs targeting ALS1 (e), and no plasmid control (f) are shown. Scale bar = 100 μ.

FIGURE 3

Targeted mutagenesis of ALS1 and EPSPS1 detected by PCR/RE and T7EI assays. Three replications of CRISPR/Cas9 and TALENs are used in the assays. (A) Schematic of PCR/restriction enzyme digestion assay (PCR/RE) and resulting gel images of (B) ALS1 and (C) EPSPS1 in which the amplicons were digested with BslI and XcmI, respectively. Mutant bands resistant to digestion in (B,C) are indicated by red arrow and were cloned for Sanger sequencing. (D) Schematic of T7 Endonuclease I assay (T7EI) and resulting gel images of (E) ALS1 and (F) EPSPS1. Arrows indicate expected cleavage products from targeted mutagenesis and indel percentage for each sample is indicated below. Sanger sequences of the targeted mutagenesis site of (G) ALS1 and (H) EPSPS1. ^∗ Denote the bands present in mutagenized samples only. Protospacer adjacent motif (PAM) is indicated in red. 100 bp NEB ladder; WT/D, wild type digested; WT/U, wild type un-digested.

FIGURE 4

Quantification of targeted mutagenesis at ALS1 locus by amplicon sequencing. (A) Percentage of mutagenized reads at ALS1 locus are shown using CRISPR/Cas9 and TALENs. The samples represent three biological replicates and three technical replicates of the PCR for each reagent, Student’s t-test (P ≤ 0.005). (B) Frequency of the types of mutations in each sample are shown. Pie charts below represents the total average for each kind of mutation.

FIGURE 5

Overview of dsODN integration method for evaluating targeted mutagenesis caused by SSNs in protoplasts. (A) Transient transformation of protoplasts with SSN plasmid constructs plus dsODNs (scale bar = 20 μ) and genomic DNA isolation. (B) Workflow for detecting SSN mediated on-target cleavage by insertion of a blunt ended dsODN at the double stranded break (DSB) site in protoplast cells and screening for qualitative assessment of functionality. (C) The dsODN with 5^′ phosphorylation and end protection by phosphorothioate linkages at both 5^′ and 3^′ ends of both strands shown in red rectangle (Tsai et al., 2015). (D) Schematic of dsODN insertion (red) in two possible directions at the target site, primers used for PCR screening along with a resulting gel image are shown. dsODN specific primers (2,3 in red are complementary to each other) and target gene specific primers (1,4 in blue) are used in combinations shown to consider directionality of dsODN integration. The expected results from the PCR reactions are presented. 1+4 reaction is a positive control. (E) Schematic of amplicon sequencing of dsODN inserted target sites. dsODN, double stranded oligodeoxynucleotide; SSN, sequence specific nuclease.

FIGURE 6

Detection of dsODNs integration in ALS1 and EPSPS1 by PCR. (A) dsODN insertion is shown at the DSB induced by SSN in target genes and arrows represent the primers used to detect the on-target activity of the dsODN (Supplementary Table S2). The order of PCR reactions for each SSN is shown (B) Gel image showing dsODN integration at ALS1 and (C) EPSPS1. Target genes have been amplified using dsODN specific primer and gene specific primer. dsODN only is wild type/negative control without nuclease but with dsODNs to account for background DSBs. PCR amplicon sizes assuming one dsODN integration are given. DSB, double stranded break; M, NEB 100 bp ladder.

FIGURE 7

Detection of dsODNs integration by PCR using AtCas9, eCas9 (1.1) and Cas9-HF1 targeting ALS1 and ALS2. Gel images showing dsODN integration at ALS locus at the DSB induced by variants of Cas9 including AtCas9, eCas9(1.1) and Cas9-HF1. (A) sg751 targets both ALS1 and ALS2 and (B) sg746 is specific for ALS1 (Supplementary Table S3). The order of PCR reactions for each SSN is according to Figure 6A. dsODN only is wild type/negative control without nuclease but with dsODNs in the protoplast transformation reactions to account for background DSB. PCR amplicon sizes assuming one dsODN integration are given which are same for all Cas9 variants. eCas9(1.1), enhanced specificity Cas9; Cas9-HF1, high fidelity Cas9. M, 100 bp NEB ladder.