High-efficiency genome editing by Cas12a ribonucleoprotein complex in Euglena gracilis

Abstract

Transgene-free genome editing based on clustered regularly interspaced short palindromic repeats (CRISPR) technology is key to achieving genetic engineering in microalgae for basic research and industrial applications. Euglena gracilis, a unicellular phytoflagellate microalga, is a promising biomaterial for foods, feeds, cosmetics and biofuels. However, methods for the genetic manipulation of E. gracilis are still limited. Here, we developed a high-efficiency, transgene-free genome editing method for E. gracilis using Lachnospiraceae bacterium CRISPR-associated protein 12a (LbCas12a) ribonucleoprotein (RNP) complex, which complements the previously established Cas9 RNP-based method. Through the direct delivery of LbCas12a-containing RNPs, our method reached mutagenesis rates of approximately 77.2-94.5% at two different E. gracilis target genes, Glucan synthase-like 2 (EgGSL2) and a phytoene synthase gene (EgcrtB). Moreover, in addition to targeted mutagenesis, we demonstrated efficient knock-in and base editing at the target site using LbCas12a-based RNPs with a single-stranded DNA donor template in E. gracilis. This study extends the genetic engineering capabilities of Euglena to accelerate its basic use for research and engineering for bioproduction.

FIGURE 1

Genome editing of Euglena gracilis using LbCas12a RNPs. (A) Overview of experimental procedures for the genome editing of E. gracilis using LbCas12a RNP complexes. (B) Design of target sequences 1 and 2 on the 5′ genomic region of EgGSL2 exon 2. (C) Representative images of cell populations at 96 h after introduction of LbCas12a RNP complexes non‐treated condition (Control) and LbCas12a RNPs targeting EgGSL2. Scale bar, 30 μm. (D) Percentage of phenotypically altered cells 96 h after introduction of LbCas12a RNP complexes in the non‐treated condition (Control) and LbCas12a RNP complexes targeting EgGSL2. Results of five independent biological replicates counting more than 1500 cells per treatment are shown as a box‐and‐whisker plot. Statistical significance was determined by ANOVA, followed by Tukey–Kramer multiple comparison test. Different letters indicate means that statistically significant differences at p < 0.01.

FIGURE 2

Efficiency of genome editing with LbCas12a RNP complexes. (A) Schematic illustration of the EgGSL2 gene model and the PCR product containing target sequences 1 and 2. (B) T7 Endonuclease I assay at 96 h after introduction of LbCas12a RNP complexes in the non‐treated condition (Control) and EgGSL2‐targeting LbCas12a RNP complexes. Arrowheads indicate the digested PCR products. M, DNA ladder marker. (C) Analysis of the InDel mutation rate at 96 h after introduction of LbCas12a RNP complexes in the non‐treated condition (Control) and EgGSL2‐targeting LbCas12a RNPs by amplicon sequencing. Unmodified indicates wild type or substitution. (D) Nucleotide distribution of the amplicon flanking the target sites at 96 h after introduction of LbCas12a RNP complexes in the non‐treated condition (Control) and EgGSL2‐targeting LbCas12a RNP complexes analysed with CRISPresso2. Black bars indicate the percentage of reads with bases deleted at that position. Brown bars between the bases indicate the percentage of reads that have an insertion at that position.

FIGURE 3

Characteristics of the isolated EgGSL2 mutant strains established by LbCas12a RNP‐based genome editing. (A) Alignment of genomic DNA sequences flanking the target sites in wild‐type and isolated EgGSL2 genome‐edited strains. (B) Representative images of the wild‐type and isolated EgGSL2 genome‐edited strains after 2 days of growth in KH medium. Scale bar, 10 μm. (C) Paramylon content in wild‐type and isolated EgGSL2 genome‐edited strains. Data are means of three independent biological replicates (n = 3) ± SD. Statistical significance was determined by ANOVA, followed by Tukey–Kramer multiple comparison test. Different letters indicate means that statistically significant differences at p < 0.01. (D) Growth curves of wild type and isolated EgGSL2 genome‐edited strains in KH medium culture. Data are means of three independent biological replicates (n = 3) ± SD.

FIGURE 4

LbCas12a RNP‐based genome editing of EgcrtB. (A) Design of the target sequence on the 5′ genomic region of EgcrtB. (B) Analysis of the InDel mutation rate at 96 h after introduction of LbCas12a RNP complexes in the non‐treated condition (Control) and EgcrtB‐targeting LbCas12a RNP complexes by amplicon sequencing. Unmodified indicates wild type or substitution. (C) Nucleotide distribution of the amplicon flanking the target site at 96 h after introduction of LbCas12a RNP complexes in the non‐treated condition (Control) and EgcrtB‐targeting LbCas12a RNP complexes analysed with CRISPresso2. Black bars indicate the percentage of reads with bases deleted at that position. Brown bars between the bases indicate the percentage of reads that have an insertion at that position. (D) Alignment of genomic DNA sequences flanking the target sites in wild‐type and isolated EgcrtB genome‐edited strains. (E) Representative images of wild‐type and isolated EgcrtB genome‐edited strains after 4 days of growth in KH medium. Upper: Images of cultured liquid. Lower: Microscopy images. Scale bar, 10 μm. (F) Chlorophyll a + b content in wild‐type and isolated EgcrtB genome‐edited strains. Data are means of three independent biological replicates (n = 3) ± SD. Statistical significance was determined by ANOVA, followed by Tukey–Kramer multiple comparison test. Different letters indicate means that statistically significant differences at p < 0.01. (G) Growth curves of wild type and isolated EgcrtB genome‐edited strains in KH medium culture. Data are means of three independent biological replicates (n = 3) ± SD.

FIGURE 5

Knock‐in at EgGSL2 target site using LbCas12a RNP complexes and ssODNs. (A) Design of the ssODN sequence for knock‐in at the EgGSL2 target site 2. (B) PCR products derived from the cell population 96 h after introduction of LbCas12a RNP complexes in the non‐treated condition (Control) and ssODNs for knock‐in and LbCas12a RNP complexes targeting EgGSL2. Arrowheads indicate the EcoRI‐digested PCR products. (C) Percentage of precise knock‐in at 96 h after introduction of LbCas12a RNP complexes in the non‐treated condition (Control), EgGSL2‐targeting LbCas12a RNPs and ssODNs by amplicon sequencing are shown as a box‐and‐whisker plot. Statistical significance was determined by Welch's t‐test. Asterisks indicate the statistically significant differences at p < 0.05. (D) PCR products derived from the wild‐type and isolated EgGSL2 knock‐in‐type genome‐edited strains. Arrowheads indicate the EcoRI‐digested PCR products. (E) Representative images of wild‐type and isolated EgGSL2 knock‐in‐type genome‐edited strains after 2 days of growth in KH medium. Scale bar, 10 μm. (F) Sanger sequencing peak data of wild‐type and isolated EgGSL2  knock‐in‐type genome‐edited strains.

FIGURE 6

Base editing at a EgGSL2 target site using LbCas12a RNPs and ssODNs. (A) Design of the ssODN sequence for base editing of the EgGSL2 target site 2. (B) PCR products derived from the cell population 96 h after introduction of LbCas12a RNP complexes in the non‐treated condition (Control) and ssODNs for base editing and LbCas12a RNP complexes targeting EgGSL2. Arrowheads indicate the HindIII‐digested PCR products. (C) Percentage of precise base‐editing at 96 h after introduction of LbCas12a RNP complexes in the non‐treated condition (Control), EgGSL2‐targeting LbCas12a RNPs and ssODNs by amplicon sequencing are shown as a box‐and‐whisker plot. Statistical significance was determined by Welch's t‐test. Asterisks indicate the statistically significant differences at p < 0.05. (D) PCR products derived from the wild‐type and isolated EgGSL2 target site base‐edited strains. Arrowheads indicate the HindIII‐digested PCR products. (E) Representative images of wild‐type and isolated EgGSL2 target site base‐edited strains after 2 days of growth in KH medium. Scale bar, 10 μm. (F) Sanger sequencing peak data of wild‐type and isolated EgGSL2 target site base‐edited strains. (G) Alignment of target sites in wild type and base‐edited strains.