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CRISPR-Cas technologies have revolutionized the field of genome editing. However, finding and isolating the desired germline edit remains a major bottleneck. Therefore, this protocol describes a robust method for quickly screening F0 CRISPR-injected zebrafish sperm for germline edits using standard PCR, restriction digest, and gel electrophoresis techniques.
The advent of targeted CRISPR-Cas nuclease technologies has revolutionized the ability to perform precise genome editing in both established and emerging model systems. CRISPR-Cas genome editing systems use a synthetic guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to specific genomic DNA loci, where the Cas endonuclease generates a double-strand break. The repair of double-strand breaks by intrinsic error-prone mechanisms leads to insertions and/or deletions, disrupting the locus. Alternatively, the inclusion of double-stranded DNA donors or single-stranded DNA oligonucleotides in this process can elicit the inclusion of precise genome edits ranging from single nucleotide polymorphisms to small immunological tags or even large fluorescent protein constructs. However, a major bottleneck in this procedure can be finding and isolating the desired edit in the germline. This protocol outlines a robust method for screening and isolating germline mutations at specific loci in Danio rerio (zebrafish); however, these principles may be adaptable in any model where in vivo sperm collection is possible.
The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas system is a powerful tool to perform loci-specific mutagenesis and precise genome editing in the Danio rerio (zebrafish) model system1,2,3,4. The Cas-ribonucleoprotein (RNP) is comprised of two main components: a Cas endonuclease (commonly Cas9 or Cas12a) and a locus-specific synthetic guide RNA (sgRNA)5. Together, the Cas-RNP generates a double-stranded break (DSB) in the desired locus that can be repaired by one of two intrinsic repa....
This study was carried out in line with the guidelines in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the University of Texas at Austin Animal Care and Use Committee (AUP-2021-00254).
1. Designing the sgRNA for CRISPR mutagenesis
The experimental approaches described in this protocol allow for the more rapid identification of genome edits or putative deleterious alleles by focusing on the analysis of thousands of genomes derived from the collection of F0-injected male sperm. Figure 2 highlights how to interpret the results obtained using this protocol.
To generate mutations in the p2ry12 locus, one-cell stage zebrafish embryos were injected with Cas9 endonuclease and a p2ry12<.......
This protocol describes a method for rapidly characterizing putative genome edits or targeted mutations using CRISPR-Cas technology by focused analysis on F0 male sperm genomes. This protocol should be amenable to other animal models where sperm is readily available for sampling without euthanasia. This method will increase the throughput of screening for desired edits and is especially useful for identifying rare HDR-mediated knock-in events. This approach also serves to reduce the number of experimental animals used to.......
We would like to thank Anna Hindes at Washington University School of Medicine for her initial efforts in obtaining good-quality sperm genomic DNA using the hot shot method. This work was funded by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award (R01AR072009 to R.S.G.).
....Name | Company | Catalog Number | Comments |
Agarose powder | Fisher BioReagents | BP1356-100 | |
Breeding tanks | Carolina Biological | 161937 | |
BstNI Restriction Enzyme | NEB | R0168S | |
Cas9 Endonuclease | IDT | 1081060 | |
DNA Ladder, 100 bp | Thermo Scientific | FERSM0241 | |
dnah10 donor construct  | Sigma-Aldrich | DNA Oligo in Tube; 0.025 nM, standard desalt purification, dry. Phosphorothioate bond on the donor at the first three phosphate bonds on both the 5’ and 3’ ends (5'-CCTCTCTCCCTTTCAGAAGCTTC TGCTCATCCGCTGCTTCTGCCT GGACCGAGTGTACCGTGCCGTC AGTGATTACGTCACGC-3') | |
dnah10 forward primer | Sigma-Aldrich | DNA Oligo in Tube; 0.025 nM, standard desalt purification, dry (5'-CATGGAACTCTTTCCTAATGAGT TTGGC-3') | |
dnah10 reverse primer | Sigma-Aldrich | DNA Oligo in Tube; 0.025 nM, standard desalt purification, dry ('5-AGTAGAGATCACACATCAACAGA ATACAGC-3') | |
dnah10 synthetic sgRNA | Synthego | Synthetic sgRNA, target sequence: 5'-GCTCATCCGCTGCTTCAGGC-3' | |
Electrophoresis power supply | Thermo Scientific | 105ECA-115 | |
Filter forceps | Millipore | XX6200006P | |
Fish (system) water | Generic | n/a | |
Gel electrophoresis system (including casting frame, comb, and electrophoresis chamber) | Thermo Scientific | B2 | |
Gel imaging light box | Azure Biosystems | AZI200-01 | |
Gel stain, 10000XÂ | Invitrogen | S33102 | |
Glass bowl, 250 mLÂ | Generic | n/a | |
Isolation tanks, 0.8 LÂ | Aquaneering | ZT080 | |
Microcap capillary tube with bulb, 20 µL | Drummond | 1-000-0020/CA | |
Minicentrifuge | Bio-Rad | 12011919EDU | |
Micropipettes, various with appropriate tips | Generic | n/a | |
Microwave | Generic | n/a | |
Nuclease free water | Promega | P119-C | |
Paper towels | Generic | n/a | |
PCR tubes, 0.2 mL | Bioexpress | T-3196-1 | |
Plastic spoon, with drilled holes/slots | Generic | n/a | |
KCl solution, 0.2 M RNAse Free | Sigma-Aldrich | P9333 | |
p2ry12 forward primer | Sigma-Aldrich | DNA Oligo in Tube; 0.025 nM, standard desalt purification, dry (5'-CCCAAATGTAATCCTGACCAGT -3') | |
p2ry12 reverse primer | Sigma-Aldrich | DNA Oligo in Tube; 0.025 nM, standard desalt purification, dry (5'-CCAGGAACACATTAACCTGGAT -3')Â | |
p2ry12 synthetic sgRNA | Synthego | Synthetic sgRNA, target sequence: 5'-GGCCGCACGAGGTCTCCGCG-3' | |
Restriction Enzyme 10X Buffer | NEB | B6003SVIAL | |
NaOH solution, 50 mM | Thermo Scientific | S318; 424330010 | |
Sponge, 1-inch x 1-inch cut with small oval divot | Generic | n/a | |
Stereomicroscope | Zeiss | Stemi 508 | |
Taq polymerase master mix, 2X | Promega | M7122 | |
TBE Buffer Concentrate, 10X | VWR | E442 | |
Thermal Cycler | Bio-Rad | 1861096 | |
Tissue paper | Fisher Scientific | 06-666 | |
Tricaine-methanesulfonate solution (Syncaine, MS-222), 0.016% in fish water (pH 7.0±0.2) | Syndel | 200-266 | |
Tris Base, 1M (Buffered with HCl to ph 8.0)Â | Promega | H5131 |
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