Versatile in vitro assay to recognize Cas9-induced mutations

Abstract

The discovery of CRISPR/Cas9 has revolutionized molecular biology, and its impact on plant biotechnology and plant breeding cannot be over-estimated. In many plant species, its application for mutagenesis is now a routine procedure--if suitable target sites, sufficient expression of the Cas9 protein, and functioning sgRNAs are combined. sgRNAs differ in their efficiency, depending on parameters that are only poorly understood. Several software tools and experience from growing databases are supporting the design of sgRNAs, but some seemingly perfect sgRNAs turn out to be inefficient or fail entirely, and most data bases stem from work with mammalian cells. Different in vitro assays testing sgRNAs in reconstituted Cas9 complexes are available and useful to reduce the risk of failure, especially in plants when CRISPR/Cas9 application requires modifications within the germ line and laborious transformation protocols. Low sgRNA efficiency and long generation times in plants can also contribute to the workload and costs of screening for the wanted genome edits. Here, we present a protocol in which a simple, initial in vitro test for suitable sgRNAs is modified to accelerate genotyping of Cas9-induced mutations. We demonstrate applicability of our protocol for mutagenesis and mutation screen for specific genes in Arabidopsis, but the principle should be universally suitable to provide a simple, low-cost, and rapid method to identify edited genes also in other plants and other organisms.

Keywords: CRISPR/Cas9; cost and labor‐saving protocol; genotyping protocol; in vitro cleavage; mutagenesis; sgRNA cleavage efficiency.

FIGURE 1

In vitro assay to estimate cleavage efficiency of sgRNA/Cas9 complexes on PCR‐amplified target DNA. (a) PCR products containing one or several sgRNA target sites (blue arrows) were amplified with flanking primers (black arrows) so that successful cleavage by a Cas9‐RNP would lead to fragments of different sizes. PCR‐generated templates should be 1–3 kb long to ensure sufficient resolution on a standard agarose gel. (b) Representative agarose gel after in vitro cleavage assays testing ten different sgRNAs (1–10) targeting the PIE1 gene of Arabidopsis thaliana. PCR templates (of four different sizes, Table S1) incubated with recombinant Cas9 protein lacking a specific sgRNA (−) were not cleaved (−); addition of the sgRNA/Cas9 complex resulted in complete (+++), moderate (++), or poor (+) cleavage. Standard concentrations of Cas9, sgRNA, and cleavage template were 30 nM, 30 nM and 3 nM, respectively, incubation time was 1 hr at 37°C. M = 1 kb marker. (c) Overview of experimental workflow

FIGURE 2

Determination of pool size for amplification of mutated alleles prior to in vitro cleavage with Cas9/sgRNA complex. (a) Location of primers and sgRNA target sites in the gene region. PIE1_3 and PIE1_4: binding sites for two sgRNAs used to mutate the PIE1 gene of Arabidopsis. PIE_TEF_and PIE_TER: PCR primers flanking both sites. (b) gDNA extracted from pools of 50 individual seedlings, in different ratios between mutant line atr2E3 and wild type (Wt), was amplified with the primers PIE_TEF and PIE_TER and the amplicons digested overnight with Cas9‐RNPs reconstituted with PIE1_3, PIE4, or ADH1_4 (control) sgRNA, prior to gel electrophoresis. (c) Overview of experimental workflow

FIGURE 3

Validation of Cas9‐RNP genotyping to identify mutants in pools of candidates. (a) Progeny from plants transformed with pDEECO::PIE1KO containing multiplexed PIE1_3 and PIE1_4 sgRNAs were grown, nine cotyledons each from five independent populations pooled and used to extract genomic DNA. These samples were incubated overnight with Cas9‐RNPs reconstituted with either PIE1_3 or PIE1_4 sgRNAs, and aliquots of 4 ng amplified with primers flanking both sites. * marks pools with amplified bands, indicating the presence of mutated alleles. Wt (Col‐0): wild‐type control for complete cleavage, atr pie1 2E3: a previously characterized line with single mutations at both sites as positive control. M = size marker. (b) Seedlings from the five populations (in the same order as in (a)) were grown on soil. Blue arrows in pool 3–1, 3–2, and 3–5 indicate plants with the pie1 phenotype (serrated leaves and stunted growth), indicating homozygous mutant alleles, frequent biallelic mutations, and congruency with the genotyping assay in (a)

FIGURE 4

Validation of Cas9‐RNP genotyping to characterize individual mutations. (a) GFP‐negative seeds in progeny of Cas9‐mutagenized plants (Table 2), indicating loss of the Cas9 transgene by segregation, were grown and genomic DNA was prepared from individual plants. This was amplified with primers flanking the mutation target sites. (b) Amplicons from (A) untreated (−) or after in vitro incubation (+) with Cas9 reconstituted with PIE1_3 (top) or PIE1_4 (bottom). **marks non‐cleaved amplicons from homozygous mutant plants, * marks partially cleaved amplicons from heterozygous plants. Wt: Col‐0 control for complete cleavage. (c) Non‐cleaved amplicons from Cas9‐RNP‐genotyped mutations in (b) were subjected to Sanger sequencing, confirming different independent mutations and homo‐ and heterozygosity. (d) Overview of experimental workflow