CRISPR-Cas9-Mediated Precise Knock-In Edits in Zebrafish Hearts

Podsumowanie

This protocol describes an approach to facilitate precise knock-in edits in zebrafish embryos using CRISPR-Cas9 technology. A phenotyping pipeline is presented to demonstrate the applicability of these techniques to model a Long QT Syndrome-associated gene variant.

Streszczenie

Clustered regularly interspaced short palindromic repeats (CRISPR) in animal models enable precise genetic manipulation for the study of physiological phenomena. Zebrafish have been used as an effective genetic model to study numerous questions related to heritable disease, development, and toxicology at the whole-organ and -organism level. Due to the well-annotated and mapped zebrafish genome, numerous tools for gene editing have been developed. However, the efficacy of generating and ease of detecting precise knock-in edits using CRISPR is a limiting factor. Described here is a CRISPR-Cas9-based knock-in approach with the simple detection of precise edits in a gene responsible for cardiac repolarization and associated with the electrical disorder, Long QT Syndrome (LQTS). This two-single-guide RNA (sgRNA) approach excises and replaces the target sequence and links a genetically encoded reporter gene. The utility of this approach is demonstrated by describing non-invasive phenotypic measurements of cardiac electrical function in wild-type and gene-edited zebrafish larvae. This approach enables the efficient study of disease-associated variants in a whole organism. Furthermore, this strategy offers possibilities for the insertion of exogenous sequences of choice, such as reporter genes, orthologs, or gene editors.

Wprowadzenie

CRISPR-based gene editing strategies in animal models enable the study of genetically heritable disease, development, and toxicology at the whole-organism level^1,2,3. Zebrafish provide a powerful model that is closer in numerous physiological aspects to humans than murine or human-derived cell models⁴. An extensive array of genetic tools and strategies have been used in zebrafish for both forward⁵ and reverse genetic screening⁶. Comprehensive genetic mapping and annotation in zebrafish have facilitated gene-editing approaches as a primary technique to engineer targeted gene knockouts (KOs) and precise knock-ins (KIs)⁷.

Despite this, generating precise KI edits in zebrafish is limited by low efficiencies and the difficulty of accurate detection. Although transcription factor-like effector nucleases (TALENs) have been successfully used and optimized for KIs⁸, CRISPR provides an improved gene-editing strategy with simpler sgRNA targeting. Numerous studies have used CRISPR to generate precise KIs in zebrafish^{9,10,11,12,13,14,15,16,17,18,19,20}, although these edits generated through CRISPR-mediated homology-directed repair (HDR) tend to be inefficient with low intrinsic success rates that require genotyping as a primary screen^9,10,14,21. This demonstrates the need for an efficient KI CRISPR system in zebrafish, as well as a reliable high-throughput system for detecting precise edits.

The goal of this study was to describe a platform for generating a precise cardiac gene KI in zebrafish hearts with simple and high-throughput detection of successful edits. A CRISPR-Cas9-based two-sgRNA exon replacement approach is described, which is based on a TALEN approach⁸. This approach involves excision of the target sequence using two-sgRNA guides and replacement with an exogenous template sequence that contains the KI of interest as well as a genetically encoded intronic reporter gene (Figure 1). The integration of a genetically encoded fluorescent reporter within the target gene intronic sequence enables the efficient detection of positive edits. A phenotyping platform is then described for assessing cardiac electrical function in zebrafish larvae for non-invasive characterization of the gene variants associated with inherited LQTS, a cardiac electrical disorder that predisposes individuals to sudden cardiac death.

These approaches will enhance the access to and use of zebrafish KI gene edits to model inherited diseases and address biological and physiological questions, such as mapping gene expression patterns, and developmental regulation. Since zebrafish hearts better parallel human cardiac electrophysiological characteristics than murine models, they may be particularly attractive as a genetically tractable system for cardiac disease modeling^7,22,23.

Protokół

Studies using zebrafish were conducted in agreement with the policies and procedures of the Simon Fraser University Animal Care Committee and the Canadian Council of Animal Care and were completed under protocol # 1264K-18.

1. Design of CRISPR components for precise edits

1. To design the two-sgRNA guides that will be used to excise the sequence containing the KI target site, first identify the zebrafish ortholog for the gene of interest.
   NOTE: Figure 2 provides a summary overview of the steps to engineer precise edits using the two-sgRNA CRISPR-Cas9 approach.
2. Next, use a design software tool, such as CRISPOR²⁴, which includes selection for Danio rerio as a species and of the Cas enzyme to be used.
   NOTE: The gene of interest for this study was zkcnh6a (Ensembl Transcript ID: ENSDART00000090809.6; UniProt Protein ID: B3DJX4), and the target mutation was amino acid, R56Q.
   1. For the two-sgRNA approach, choose one sgRNA location that precedes the target exon and a second sgRNA that is located within the immediate downstream intron.
   2. Ensure that the selected sgRNAs have high specificity and low predicted off-target binding. Use CRISPOR rankings to identify guides with minimal off-target binding. Do not consider guides with no mismatches in the seed sequence of potential off-targets.
   3. Identify the most likely potential off-target sites (based on CRISPOR scores, select the top three exon potential off-target sites) for PCR-based Sanger sequencing genotyping in step 6.1.3.
   4. Once the two-sgRNAs have been selected, obtain the reverse complement for each, such that there are four oligonucleotides to be used: two complementary oligos that precede the mutation and two complementary oligos that are downstream.
   5. On each of the four oligos, add compatible restriction sites for incorporation into a guide plasmid of choice; for integration into the DR274 plasmid, use a 5' BsaI restriction site to create an overhang. Ensure that the Bsa1 recognition site is engineered at the 5' end of the guide selected from CRISPOR and the Bsa1 recognition site is engineered at the 5' end of the complementary strands to ensure correct orientation of the guides in the DR274 plasmid (see Figure 3).
3. To design the exogenous template used for HDR in zebrafish (Figure 1), choose two sequence fragments that will flank the mVenus YFP reporter gene housed in the pKHR5 plasmid.
   NOTE: The intended modification/edit can be included in either the upstream or downstream fragment.
   1. Using Ensembl, locate the target site within the gene sequence of interest, including approximately the 2 kb flanking sequence (homology arm), which will be used to make the template.
      NOTE: Homology arms may be symmetrical or asymmetrical^25,26 and approximately 1 kb each, upstream and downstream of the target site.
   2. Divide the template into two segments that will be inserted either side of the mVenus YFP reporter gene (see Figure 4). Ensure that the split site is in an intron so that the coding sequence is not interrupted.
      NOTE: If the gene is well characterized, check for functional roles in the intron, such as splicing sites or regulatory regions. Regions close to the 5' or 3' ends are more often involved in mRNA splicing.
   3. Incorporate modifications into the template sequence that include i) silent mutations in the guide protospacer adjacent motif (PAM) or seed sequence (be aware of alternate PAM sites that the Cas enzyme might target) to prevent Cas enzyme recutting; ii) the modification of interest; iii) the creation of restriction endonuclease sites to facilitate cloning into the pKHR5 plasmid, which contains the mVenus YFP reporter gene (see Figure 3).
      NOTE: In this study, the first template segment contained XhoI upstream of the R56Q mutation site and SalI downstream, while the second template segment had EcoRI upstream of the guide target sequence and BamHI downstream. If any of the selected restriction sites are present within the template sequence, mutations to silence these will be required, or alternate approaches such as Gibson Assembly can be used.

2. Preparation of CRISPR components for embryo microinjection

1. Prepare the Cas9 for microinjection 1 week prior to the microinjections. Use Cas9 protein or prepare Cas9 mRNA via in vitro transcription.
   NOTE: In this study, Cas9 mRNA was used, since efficiencies tended to be higher.
   1. Amplify bacterial cultures of commercially available XL1 Blue bacterial agar stab (containing Cas9 plasmid) using an appropriate antibiotic such as ampicillin. Use 675 µL of liquid culture (with 325 µL of glycerol) to create a backup glycerol stock for long-term storage at −80 °C.
   2. Use the remainder of the liquid culture for a miniprep purification, according to the protocol provided with the miniprep kit. Resuspend the final purified DNA in 50 µL of the provided elution buffer. Quantify the product via a spectrophotometer to examine the yield and purity.
   3. Linearize 2 µg of the purified DNA via restriction digest using an appropriate restriction enzyme and using the appropriate buffer and incubation time as listed for the enzyme of interest.
   4. Purify the linearized plasmid using a PCR Purification Kit, resuspending it in 30 µL of the provided elution buffer.
   5. After quantifying the product, use this as a template for in vitro transcription using the appropriate transcription kit for the promoter of interest. Follow the provided protocol and purify via lithium chloride precipitation²⁷. Resuspend the purified RNA in 10 µL of nuclease-free H₂O and quantify it before storing at −20 °C for use in the microinjection mix.
2. Prepare the two sgRNA guides.
   1. Prepare the sgRNA plasmid with a scaffold by amplifying bacterial cultures from the commercially available XL1 Blue bacterial agar stab (see Table of Materials for details) in the same way as MLM3613 above (step 2.1.1), except use kanamycin instead of ampicillin.
   2. Anneal the two pairs of complementary single-stranded oligonucleotides (ssODNs) for the sgRNA guides designed above by first resuspending the ssODNs in 1x annealing buffer to a concentration of 100 µM.
   3. In separate reactions for each of the two sgRNAs, anneal the pair of complementary ssODNs using a thermal cycler. Mix 2 µg of each complementary ssODN pair with 50 µL of annealing buffer and incubate at 95 °C for 2 min, then cool to 25 °C over 45 min.
   4. Digest a commercially available plasmid that contains a gRNA scaffold. Digest 2 µg of the DR274 plasmid using 1 µL of BsaI, 2 µL of appropriate buffer, and ddH₂O to 20 µL at 37 °C for 1 h. Confirm linearization (optional: purify using a PCR Purification Kit) using gel electrophoresis²⁸.
   5. In two separate ligation reactions (one for each sgRNA), ligate the annealed ssODNs with the linearized DR274 plasmid. Use a molar insert:vector ratio of 3:1, calculating the appropriate mass through an online ligation calculator. Mix the required mass of the insert and vector with 1 µL of T4 DNA ligase, 2 µL of ligation buffer, and ddH₂O to 12 µL and incubate at room temperature for 12 h.
   6. Transform 2 µL of the ligated product into appropriate competent cells (such as 10β cells) using standard approaches, and then amplify and purify the product using a commercially available Miniprep Kit. Optional: create a glycerol stock of this product.
   7. Transcribe the two sgRNAs by linearizing 2 µg of each guide using a 3' downstream restriction site that is as close to the end of the space sequence as possible. For the DR274 plasmid, linearize with HindIII, and then purify the RNA template using a PCR Purification Kit, resuspending in 30 µL of the elution buffer.
   8. Transcribe the two guides using an RNA transcription kit. Follow the manufacturer's protocol and purify via lithium chloride precipitation²⁷. Resuspend the two purified sgRNA guides in 10 µL of nuclease-free H₂O, quantify, and store at −20 °C for use in the microinjection mix.
      NOTE: The RNA transcription kit cannot incorporate a 5' cap or poly-A tail.
3. Prepare the double-stranded, exogenous HDR reporter template.
   NOTE: The template is synthesized in two parts, one upstream and one downstream of the mVenus YFP reporter gene. These two segments are synthetic constructs ordered through a commercial provider and then ligated into the pKHR5 (which contains mVenus YFP) plasmid sequentially.
   1. Prepare the pKHR5 plasmid by amplifying bacterial cultures from the commercially available DH5α bacterial strain (see Table of Materials for details) in the same way as MLM3613 above (step 2.1.1).
      NOTE: The pKHR5 plasmid contains the mVenus YFP reporter gene sequence.
   2. Resuspend the two template segments designed above to 100 µM in TE buffer and then transform into 10β cells.
   3. Digest the first template segment (the one upstream of the mVenus YFP reporter gene) and the pKHR5 plasmid using the restriction enzymes selected in step 1.3.3.
      NOTE: Sequential digest of pKHR5 is necessary due to the close proximity of the selected restriction sites in the MCS.
   4. To prepare the pKHR5 plasmid for the upstream template segment, digest 4 µg of pKHR5 (as per step 2.2.4) with SalI and then purify using a PCR purification kit. Resuspend in 30 µL of ddH₂O and use this as a template for the second digestion reaction with XhoI. Purify the product using the PCR Purification Kit.
   5. Prepare the first template segment by digesting 2 µg of the template segment (step 2.3.2), 1 µL of XhoI, 1 µL of SalI, 2 µL of appropriate buffer, and ddH₂O to 20 µL for 1 h at 37 °C and gel-purify the product.
   6. Ligate the upstream template segment from step 2.3.5 into the prepared pKHR5 using the reaction conditions described in step 2.2.5. Transform the ligated product into competent 10β cells, amplify, and purify using a miniprep (optional: create a glycerol stock of this product).
   7. Use the ligated product from step 2.3.6 (which contains the first template segment ligated into pKHR5 plasmid upstream of the mVenus YFP reporter gene) and digest to prepare for the second (downstream of the mVenus reporter) template segment. Digest 4 µg of the ligated product (from step 2.3.6, as in step 2.2.4) with BamHI, and then purify using a PCR purification kit. Resuspend in 30 µL of ddH₂O and use this as the template for the second digest reaction with EcoRI. Purify the product using the PCR Purification Kit.
   8. Prepare the second template segment by digesting 2 µg of the template segment (step 2.3.2), 1 µL of BamHI, 1 µL of EcoRI, 2 µL of appropriate buffer, and ddH₂O to 20 µL for 1 h at 37 °C and gel-purify the product.
   9. Ligate the downstream template segment into the prepared pKHR5 (from step 2.3.7) using the reaction conditions described in step 2.2.5. Transform the ligated product into competent cells, amplify, and purify using a miniprep kit. Create a glycerol stock of this final product, which contains both template segments ligated either side of the mVenus YFP reporter gene within pKHR5.
4. Prepare the microinjection mix using the Cas9 mRNA, two sgRNAs, and the exogenous HDR reporter template.
   1. Mix 200 ng/µL of Cas9 mRNA, 100 ng/µL of each sgRNA, and 200 ng/µL of exogenous HDR reporter template in 1x injection buffer to a final volume of 20 µL.
   2. Store the microinjection mix at −20 °C and discard the unused mix after three freeze-thaw cycles.
      NOTE: Use 4 nL of this microinjection mix for microinjection into the yolk sac of each embryo.

3. Breeding of zebrafish and embryo microinjection

NOTE: Protocols for zebrafish breeding and the microinjection of single-cell embryos have been described previously^29,30,31.

1. For breeding, use zebrafish of the AB strain and that are 6-12 months of age. Inject the embryos in the one-cell stage at approximately 40 min post fertilization (see Figure 5).
   NOTE: The biological sex of the injected embryos was not known; sexual dimorphism is not evident until approximately 3 months of age³².

4. Reporter gene screening of CRISPR-Cas9-edited larval zebrafish

1. Visualize YFP integration in zebrafish larvae following the microinjection of CRISPR-Cas9 components to screen for successful HDR edits.
   1. In a 25 mm Petri dish, anesthetize 24 zebrafish larvae at 3 days post fertilization (dpf) in 0.3% tricaine methane sulfonate (MS-222, buffered to pH 7.0-7.4 with HEPES and sodium hydroxide) until they lose their self-righting reflex (typically 1-2 min). Once anesthetized, transfer each larva into an individual well of a 24-well plate.
   2. Using a microscope capable of detecting GFP/YFP, screen for reporter gene fluorescence in the eyes of each individual larva.
   3. Capture images of each larva and document the presence or absence of reporter gene expression.

5. Phenotyping of CRISPR-Cas9-edited larval zebrafish

1. Following reporter gene screening, perform cardiac phenotyping (heart rate, pericardial dimensions, ECG) on each larva. Phenotype an equal number of reporter gene-positive and -negative larvae.
   1. Use a CCD camera (e.g., blackfly USB3) and video and image recording software (e.g., Micromanager for ImageJ) to measure heart rate and pericardial dimensions while the larvae are anesthetized.
   2. To measure the heart rate, using Micromanager, create a region of interest (ROI) so as to capture the heart and exclude other structures.
      1. Import the video into ImageJ as an image sequence, and ensure the correct number of frames is entered under number of images.
      2. After the file is open, use the rectangle selection tool to draw an ROI within the heart but excluding other moving elements, and save the ROI in the ROI manager (analyze | tools | ROI manager).
      3. Click plugins | install, and select the heart rate algorithm to install the code in the default folder, then select the plugin at the bottom of the plugins tab. Record the beats per minute (bpm) from the pop-up window.
         NOTE: Image detection algorithms were custom-written to detect heart rate by measuring individual pixel density changes associated with ventricular systolic contraction. The code can be found at https://github.com/dpoburko/zFish_HR.
   3. Measure pericardial dimensions using a free tool such as ImageJ to free-draw ROIs around the pericardial sac and one of the eyes. Open the image in ImageJ and use the polygon selection tool to draw an ROI first around the pericardial sac, saving it in the ROI manager as in step 5.1.2, and repeat for the eye. Select these two ROIs in the ROI manager, then click measure. Record the area for each to later calculate the area of the pericardial sac normalized to the eye area in each larva.
   4. Following heart rate and pericardial measurements, record ECG from individual larvae.
      NOTE: Protocols for recording zebrafish ECG have been described previously^33,34,35,36.

6. Genotyping of CRISPR-Cas9-edited larval zebrafish

1. Following phenotypic analyses, conduct on- and potential off-target genotyping to confirm accurate and precise HDR gene editing.
   1. Anesthetize each 3 dpf larva in 0.3% MS-222 and tail clip to isolate gDNA using the HOTShot method³⁷. Incubate each excised tail clip in 15 µL of 25 mM NaOH at 95 °C for 20 min. Then, neutralize with 1.5 µL of Tris-HCl and centrifuge at 13,800 x g for 30 s. Retain the supernatant, which contains extracted gDNA.
   2. Recover the larvae in E3 media and return them to the housing system if further development or study is intended.
   3. Using the extracted gDNA as a template, perform PCR-based Sanger sequencing of on-target and potential off-target sites.
      NOTE: Optional: a nested PCR approach may be beneficial for some gene regions.
   4. Ensure that the on-target primer design captures the mutation site and the closest sgRNA binding site. Design a separate sequencing primer to detect the transition from the inserted homology arm and the target gene to confirm integration into the gene of interest. Design primers to sequence the top three potential off-target sites identified in step 1.2.3.
      NOTE: Guide-design software programs often suggest primers to use, but customization may be necessary to achieve optimal results.
   5. Compile on- and off-target genotyping, heart rate, pericardial dimensions, ECG phenotyping, and reporter gene data identifiable for each zebrafish.

Wyniki

The successful use of this two-sgRNA exon replacement CRISPR approach is highlighted by the introduction and simple detection of a precise edit to engineer the LQTS-associated variant, R56Q, in the zkcnh6a gene in zebrafish. Figure 6 shows a representative 3 dpf larvae injected at the one-cell embryo stage with CRISPR components as described above. Figure 6A shows the presence of the YFP mVenus reporter gene expression in the eye lens as a positive repo...

Dyskusje

The engineering of precise gene edits using CRISPR-Cas9 is challenged by the low efficiencies of HDR mechanisms and their efficient detection. Here, a CRISPR-Cas9-based two-sgRNA exon replacement approach is described that produces precise edits in zebrafish with straightforward visual detection of positive edits. The efficacy of this approach is demonstrated by generating precise edits in the zkcnh6a gene. This paper shows how cardiac function in gene-edited zebrafish larvae may be assessed using non-invasive p...

Materiały

Name	Company	Catalog Number	Comments
Program
CRISPOR	TEFOR Infrastructure
ENSEMBL	European Bioinformatics Institute
ImageJ	National Institutes of Health (NIH)
Micro-Manager	Open Source (Github)
NEBiocalculator	New England Biolabs (NEB)
EQUIPMENT
24-well Plate	VWR
25 mm Petri Dish	VWR
Blackfly USB3 Camera	Teledyne FLIR
C1000 Thermal Cycler	Bio-Rad
Centrifuge 5415C	Eppendorf
EZNA Gel Extraction Kit	Omega Biotek
MAXIscript T7 Transcription Kit	Invitrogen
MaxQ 5000 Incubator	Barnstead Lab Line
Miniprep Kit	Qiagen
mMessage mMachine T7 Ultra Transcription Kit	Invitrogen
ND1000 Spectrophotometer	Nanodrop
PCR Purification Kit	Qiagen
PLI 100A Picoinjector	Harvard Apparatus
PowerPac Basic Power Supply	Bio-Rad
Stemi 305 Steroscope	Zeiss
Wide Mini Sub Cell GT Electrophoresis System	Bio-Rad
ZebTec Zebrafish Housing System	Tecniplast
SERVICES
Gene Synthesis	Genewiz
Sanger Sequencing	Genewiz
REAGENTS
10β Competent Cells	NEB
10X PCR Buffer	Qiagen
100 mM Nucleotide Mixture	ABM
Ampicillin	Sigma
BamHI Endonuclease w/ buffer	NEB
BsaI Endonuclease w/ buffer	NEB
DR274 Plasmid (XL1 Blue bacterial agar stab)	Addgene
EcoRI Endonuclease w/ buffer	NEB
Glycerol
HEPES	Sigma
HindIII Endonuclease w/ buffer	NEB
Kanamycin	Sigma
Methylene Blue	Sigma
MLM3613 Plasmid (XL1 Blue bacterial agar stab)	Addgene
MS-222 (Tricaine)	Sigma
pKHR5 Plasmid (DH5α bacterial agar stab)	Addgene
PmeI Endonuclease w/ buffer	NEB
SalI Endonuclease w/ buffer	NEB
Sodium Hydroxide	Sigma
T4 Ligase w/ buffer	Sigma
Taq Polymerase	Qiagen
TE Buffer	Sigma
Tris Hydrochloride	Sigma
XhoI Endonuclease w/ buffer	NEB
RECIPES
Solution	Component	Supplier
Annealing Buffer (pH 7.5-8.0)	10 mM Tris	Sigma
	50 mM NaCl	Sigma
	1 mM EDTA	Sigma
E3 Media (pH 7.2)	5 mM NaCl	Sigma
	0.17 mM KCl	Sigma
	0.33 mM CaCl2	Sigma
	0.33 mM MgSO4	Sigma
Injection Buffer (pH 7.5)	20 mM HEPES	Sigma
	150 mM KCl	Sigma