Wierson et al., 2019 - Expanding the CRISPR Toolbox with ErCas12a in Zebrafish and Human Cells. The CRISPR journal   2(6):417-433 Full text @ CRISPR J

FIG. 1.

Characterization and activity of CRISPR-ErCas12a in zebrafish. (A) Phylogenetic relationship between known CRISPR associated proteins and ErCas12a. Evaluation of branching was ensured by bootstrap statistical analysis (1,000 replications). (B) Workflow showing dual NLS ErCas12a mRNA and pre-crRNA injection into single-cell animals. Animals are heat shocked for 4 h and then allowed to develop normally until DNA is isolated and analyzed. (C) Schematic and sequences of pre-crRNA used to target noto.(D) Schematic and sequences of pre-crRNA used to target cx43.4. (E) Results displaying the wild-type and top five mutated alleles after AmpliconEZ analysis of two noto pre-crRNAs and one cx43.4 pre-crRNA.

FIG. 2.

Using noto:RFP-DR48 to assay the propensity of ErCas12a and SpCas9 to elicit strand annealing in zebrafish. (A) Schematic of noto:RFP-DR48 showing the location of the 48 bp direct repeats flanking both the SpCas9 and ErCas12a universal RNA cursor sites (underline) and protospacer adjacent motif (PAM) sites (red text). (B) Data plot showing the ratio of injected animals displaying red fluorescent protein (RFP) in the notochord out of the total carrying the transgene. Data plot represents the mean ± SD. p-Values calculated with one-tailed Student's t-test. (C) Qualitatively scored ratios of notochord converted to RFP+ after SpCas9 induced strand-annealing mediated repair (SAMR). (D) Qualitatively scored ratios of notochord converted to RFP+ after ErCas12a induced SAMR. (E–G and E'-G') Representative embryos for broad, intermediate, and narrow conversion of RFP in the notochord.

FIG. 3.

Targeting noto with GeneWeld. (A) Schematic of noto showing designed homology for precise integration using ErCas12a and the U-pre-crRNA. Green is designed 5′ homology. Blue is designed 3′ homology. The PAM for ErCas12a targeting in the genome and donor is underlined. (B and B′) Confocal Z-stack image showing broad green fluorescent protein (GFP) expression in the embryo. Scale bar is 100 μm. (C) Data plot showing the ratio of embryos with GFP expression in the notochord out of total injected embryos. Data plot represents the mean ± SD. (D) Gel showing 5′ junction fragment expected after precise integration using GeneWeld. (E) Gel showing 3′ junction fragment expected after precise integration using GeneWeld. (F) DNA sequencing of lane 5 in (D) and (E) showing a precise integration using the programmed homology.

FIG. 4.

ErCas12a activity in human HEK293T cells. (A) Schematic showing the workflow for ErCas12a and pre-crRNA expression in human cells from a cis expression plasmid. After transfection, cells are recovered and then analyzed. (B) Schematic of two pre-crRNAs designed to target AAVS1. (C) T7E1 assay showing AAVS1-pre-crRNA1 is active. Arrowheads are cleaved bands, indicating nuclease activity. (D) Schematic of four pre-crRNAs designed to target CCR5. (E) Schematic of four pre-crRNAs designed to target TRAC. (F) Graph showing percentage of indels after using pre-crRNAs to target AAVS1, CCR5, and TRAC in HEK293T cells as determined by ICE analysis. Data plot represents the mean ± SD. (G) Results displaying the wild-type and top five mutated alleles after AmpliconEZ analysis of AAVS1 targeted DNA.

FIG. 5.

Using pMini-CAAGs::RFP-DR48 to assay the propensity of ErCas12a and SpCas9 to elicit strand annealing in HEK293T cells. (A) Schematic showing the workflow for determining SAMR in human cells. An all-in-one expression plasmid for ErCas12a or SpCas9 is transfected along with pMini-CAAGs::RFP-DR48. Cells are allowed to recover and are then isolated with fluorescence-activated cell sorting for RFP+ cells. (B) Quantification of RFP+ cells per total cells analyzed by flow cytometry. Data plot represents the mean ± SD. p-Values calculated with one-tailed Student's t-test.

FIG. 6.

Using ErCas12a for targeted integration in human cells. (A) Schematic showing the workflow for targeted integration at AAVS1 in HEK293T cells. (B) Schematic of AAVS1 showing designed homology for precise integration using ErCas12a and the U-pre-crRNA to liberate the cassette. Green block, 5′ homology; blue block, 3′ homology. The PAM for ErCas12a targeting in the genome and donor is underlined. (C) Schematic for flow analysis after transfection of GeneWeld reagents for targeting AAVS1. (D) Bar graph showing the ratio of cells with GFP expression as determined by flow cytometry. Graph represents the mean ± SD. p-Values calculated with one-tailed Student's t-test. (E) Junction fragment gel showing the expected 5′ integration amplicon. No amplicon is seen when transfecting donor alone. (F) DNA sequencing of lanes in (E) showing a precise integration using the programmed homology.

Acknowledgments:
ZFIN wishes to thank the journal The CRISPR journal for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ CRISPR J