A PCR Based Protocol for Detecting Indel Mutations Induced by TALENs and CRISPR/Cas9 in Zebrafish

<div><p>Genome editing techniques such as the zinc-finger nucleases (ZFNs), transcription activator-like effecter nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system Cas9 can induce efficient DNA double strand breaks (DSBs) at the target genomic sequence and result in indel mutations by the error-prone non-homologous end joining (NHEJ) DNA repair system. Several methods including sequence specific endonuclease assay, T7E1 assay and high resolution melting curve assay (HRM) etc have been developed to detect the efficiency of the induced mutations. However, these assays have some limitations in that they either require specific sequences in the target sites or are unable to generate sequencing-ready mutant DNA fragments or unable to distinguish induced mutations from natural nucleotide polymorphism. Here, we developed a simple PCR-based protocol for detecting indel mutations induced by TALEN and Cas9 in zebrafish. We designed 2 pairs of primers for each target locus, with one putative amplicon extending beyond the putative indel site and the other overlapping it. With these primers, we performed a qPCR assay to efficiently detect the frequencies of newly induced mutations, which was accompanied with a T-vector-based colony analysis to generate single-copy mutant fragment clones for subsequent DNA sequencing. Thus, our work has provided a very simple, efficient and fast assay for detecting induced mutations, which we anticipate will be widely used in the area of genome editing.</p></div

Identification of mutations induced by <i>ldlr</i> TALEN.

<p>(A) A schematic diagram showing primers for detecting mutations in TALEN target sites of <i>ldlr</i> gene. (B) The relative amplification efficiency of <i>ldlr</i> Pf primers to Po primers. <i>ldlr</i> TALEN mRNAs injection dramatically reduced the amplification efficiency of <i>ldlr</i> Pf primers. 5 embryos from the wild-type group or the <i>ldlr</i>-TALEN-injected group were pooled and analyzed by qPCR, and 3 pools of each group were analyzed in an independent experiment. Similar results were obtained in three independent experiments. (C) Identification of a mutation in a single allele with T-CIA. Upper panel was an agarose electrophoresis result of PCR products amplified with <i>ldlr</i> Po and Pf primers. Lower panel listed the sequences of mutant colonies identified in upper panel.</p

Schematic diagrams showing a PCR-based protocol for identifying mutations induced by TALEN and Cas9 in zebrafish.

<p>(A) Primers designed for detecting mutations in target site. (B) Procedure for identifying induced mutation in zebrafish. WT, wild-type. MT, mutant.</p

Identification of mutations induced by <i>nsd2</i> Cas9.

<p>(A) A schematic diagram showing primers for detecting mutations in Cas9 target site of <i>nsd2</i> gene. (B) The relative amplification efficiency of <i>nsd2</i> Pf primers to Po primers. <i>nsd2</i> Cas9 mRNA injection dramatically reduced the amplification efficiency of <i>nsd2</i> Pf primers. 5 embryos from the wild-type or the <i>nsd2</i>-Cas9-injected group were pooled and analyzed by qPCR, and 3 pools of each group were analyzed in an independent experiment. Similar results were obtained in three independent experiments. (C) Identification of a mutation in a single allele with T-CIA. Upper panel was an agarose electrophoresis result of PCR products amplified with <i>nsd2</i> Po and Pf primers. Lower panel listed the sequences of mutant colonies identified in upper panel.</p

Determination of known mutations with qPCR.

<p>(A) Relative levels of Po or Pf PCR products. The amounts of Po or Pf PCR products of mixed templates were compared to the amount of corresponding PCR products of pure wild-type template. (B) Relative ratios between Po PCR products and Pf PCR products. The ratios between Po PCR products and Pf PCR products of mixed templates were compared to the ratio of wild-type template. WT, wild-type. MT, mutant.</p

Fast skeletal muscle expresses ectopic endothelial genes following Etv2 overexpression.

<p>(A) Immunostained sections through the trunk of 48 hpf <i>hsp70l:etv2/fli1a:EGFP</i> embryos that were untreated (control) or heat shocked at 24 hpf (HS+24 h). Sections were stained for GFP and fast muscle myosin. Nuclei are stained with DAPI in the mergeDAPI panels. <i>fli1a:EGFP</i> is normally expressed in the intersomitic vessels (ISVs) and axial vessels (AVs) of control sections. However, following heat shock, many fast muscle myosin positive cells were also GFP positive (A). ROI is the region of interest highlighted by the dashed box in each panel. One section from 20 different embryos was observed for each treatment group with similar results within each group. (B) Confocal projection images of a <i>kdrl:GFP</i>⁺ and <i>mylpfa:mRFP</i>⁺ double positive muscle fiber (arrow) in a living embryo 12 h post–heat shock. (C) Time lapse imaging of the trunk (left column) and at the single cell level (right column) of a <i>mylpfa:mRFP/hsp70l:etv2/kdrl:GFP</i> triple transgenic embryo beginning at 8 h post–heat shock (t₀+8 h). Heat shock occurred at 24 hpf. A few Etv2-mCherry⁺ nuclei are present in the first panel (arrowhead). The normal GFP⁺ intersomitic vessels (ISVs) and axial vessels (AVs) are labeled. <i>mylpfa:mRFP</i> labels fast muscle fibers in red. In the trunk, GFP expression first appears in muscle fibers between t₀+8 h to t₀+10 h and progresses in an caudal to rostral wave. mRFP⁺ fibers induce GFP expression and then soon switch off mRFP expression. ISV sprouts appear to apoptose and regress (asterisks). At the single cell level, mRFP⁺ fibers become GFP⁺ and then change morphology, a single cell is highlighted by a dashed outline in the right column.</p

Transdifferentiation of Fast Skeletal Muscle Into Functional Endothelium in Vivo by Transcription Factor Etv2

<div><p>Etsrp/Etv2 (Etv2) is an evolutionarily conserved master regulator of vascular development in vertebrates. Etv2 deficiency prevents the proper specification of the endothelial cell lineage, while its overexpression causes expansion of the endothelial cell lineage in the early embryo or in embryonic stem cells. We hypothesized that Etv2 alone is capable of transdifferentiating later somatic cells into endothelial cells. Using heat shock inducible Etv2 transgenic zebrafish, we demonstrate that Etv2 expression alone is sufficient to transdifferentiate fast skeletal muscle cells into functional blood vessels. Following heat treatment, fast skeletal muscle cells turn on vascular genes and repress muscle genes. Time-lapse imaging clearly shows that muscle cells turn on vascular gene expression, undergo dramatic morphological changes, and integrate into the existing vascular network. Lineage tracing and immunostaining confirm that fast skeletal muscle cells are the source of these newly generated vessels. Microangiography and observed blood flow demonstrated that this new vasculature is capable of supporting circulation. Using pharmacological, transgenic, and morpholino approaches, we further establish that the canonical Wnt pathway is important for induction of the transdifferentiation process, whereas the VEGF pathway provides a maturation signal for the endothelial fate. Additionally, overexpression of Etv2 in mammalian myoblast cells, but not in other cell types examined, induced expression of vascular genes. We have demonstrated in zebrafish that expression of Etv2 alone is sufficient to transdifferentiate fast skeletal muscle into functional endothelial cells in vivo. Given the evolutionarily conserved function of this transcription factor and the responsiveness of mammalian myoblasts to Etv2, it is likely that mammalian muscle cells will respond similarly.</p></div

Etv2 cell autonomously initiates transdifferentiation of muscle cells.

<p>(A) Blastula cell transplantation was performed from triple transgenic, <i>mylpfa:mRFP/hsp70l:etv2/kdrl:GFP⁺</i>, into wild-type embryos. Approximately 10 cells were transplanted per embryo. Transplanted embryos were raised until 22 hpf, at which point they were selected for embryos displaying <i>mylpfa:mRFP</i> expression in distinct regions absent in <i>kdrl:GFP</i>, region of interest (ROI) boxed in (B) corresponds to images in (C). These embryos were then either heat shocked or left as no heat shock controls. Embryos were then analyzed for <i>mylpfa:mRFP/kdrl:GFP</i> coexpression at 10 h post–heat shock and followed out to 42 h post–heat shock (C). A muscle cell labeled with the arrow undergoes transdifferentiation to form a lumenized vessel (C). (D) Quantification of transdifferentiation efficiency per muscle cell. Only clearly distinguishable muscle cells were counted. Thirty-eight chimeric embryos, 312 total cells, were observed in the heat-shocked condition, and 20 chimeric embryos, 143 total cells, were observed for the control non–heat shocked condition.</p

VEGF signaling is dispensable for induction but necessary for maturation of Etv2 induced vasculature.

<p>(A–D) Control <i>kdrl:GFP</i> embryos (A) or <i>kdrl:GFP/hsp70l:etv2</i> (B–D) embryos following heat shock at 24 hpf and imaged at 24 h or 36 h post–heat shock. Control embryos exhibit normal vascular <i>kdrl:GFP</i> expression (A), while <i>kdrl:GFP/hsp70l:etv2</i> embryos exhibit the ectopic GFP and morphological changes previously described (B–D). (E–H) VEGFAa morpholino (VEGF-MO) treated embryos lack intersomitic vessels (E) but still induce <i>kdrl:GFP</i> in muscle fibers (F). However, <i>kdrl:GFP</i>⁺ muscle fibers do not undergo the normally observed morphological changes following heat shock–induced expression of Etv2 (G,H). (I–L) Overexpression of VEGFAa¹²¹ (VEGF OE) driven by the <i>hsp70l</i> promoter results in disorganization and expansion of the normal vasculature (I). Following heat shock–induced expression of Etv2, no significant change in the number of muscle fibers expressing <i>kdrl:GFP</i> is observed (J). However, the morphological changes observed are accelerated in the presence of elevated VEGF (K,L). (M–P) Treatment of embryos with SU5416, a Kdr inhibitor, similarly inhibits intersomitic vessel development in control embryos (M). However, drug treatment does not inhibit induction of <i>kdrl:GFP</i> following heat shock–induced Etv2 expression in muscle (N). The morphology and survival of these fibers is compromised when Kdr is inhibited (O,P). (Q–V) Removal of VEGF inhibitor SU5416 24 h following heat shock allows for survival and maturation of transdifferentiated cells. <i>Kdrl:GFP/hsp07l:etv2</i> embryos were heat shocked at 22 hpf and then treated with DMSO carrier or SU5416 for 24 h at which point the drug was either maintained (S,T) or removed (U,V) and the embryos were allowed to develop until 72 hpf. (Q,R) DMSO controls exhibit a transdifferentiated vascular network similar to that in untreated controls. (S,T) Sustained SU5416 treatment largely abolishes the <i>kdrl:GFP⁺</i> vascular network. (U,V) Removal of SU5416 24 h post–heat shock results in the development of a vascular network similar to controls (Q,R), suggesting VEGF signaling modulates the survival and maturation of muscle-derived vessels and not the initial induction. For all experiments at least 20 embryos were observed with similar results.</p