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May 2020

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GeneWeld: Efficient Targeted Integration Directed by Short Homology in Zebrafish
GeneWeld:斑马鱼短同源性诱导的高效靶向整合   

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Abstract

Efficient precision genome engineering requires high frequency and specificity of integration at the genomic target site. Multiple design strategies for zebrafish gene targeting have previously been reported with widely varying frequencies for germline recovery of integration alleles. The GeneWeld protocol and pGTag (plasmids for Gene Tagging) vector series provide a set of resources to streamline precision gene targeting in zebrafish. Our approach uses short homology of 24-48 bp to drive targeted integration of DNA reporter cassettes by homology-mediated end joining (HMEJ) at a CRISPR/Cas induced DNA double-strand break. The pGTag vectors contain reporters flanked by a universal CRISPR sgRNA sequence to liberate the targeting cassette in vivo and expose homology arms for homology-driven integration. Germline transmission rates for precision-targeted integration alleles range 22-100%. Our system provides a streamlined, straightforward, and cost-effective approach for high-efficiency gene targeting applications in zebrafish.


Graphic Abstract:



GeneWeld method for CRISPR/Cas9 targeted integration.


Keywords: CRISPR/Cas9 (CRISPR/Cas9), Knock-in (基因敲入), Homology mediated-end joining (同源介导端连接), Targeted integration (靶向整合), Zebrafish (斑马鱼)

Background

Designer nucleases have rapidly expanded the way in which researchers can utilize endogenous DNA repair mechanisms for creating gene knock-outs, reporter gene knock-ins, gene deletions, single nucleotide polymorphisms, and epitope-tagged alleles in diverse species (Beumer et al., 2008; Geurts et al., 2009; Bedell et al., 2012; Carlson et al., 2012; Yang et al., 2013). A single dsDNA break in the genome results in increased frequencies of recombination and promotes integration of homologous recombination (HR)-based vectors (Orr-Weaver et al., 1981; Hasty et al., 1991; Rong and Golic, 2000; Zu et al., 2013; Shin et al., 2014; Hoshijima et al., 2016). Additionally, in vitro or in vivo linearization of targeting vectors stimulates homology-directed repair (HDR) (Orr-Weaver et al., 1981; Jasin and Berg, 1988; Hasty et al., 1991; Rong and Golic, 2000; Zu et al., 2013; Shin et al., 2014; Hoshijima et al., 2016). Utilizing HDR or HR at a targeted double-strand break (DSB) allows directional knock-in of exogenous DNA with base-pair precision; however, reported frequencies vary widely, and engineering targeting vectors with long homology arms is not straightforward.


Previous work has shown that Xenopus oocytes have the ability to join or recombine linear DNA molecules that contain short regions of homology at their ends, and this activity is likely mediated by exonuclease activity, allowing base pairing of the resected homology (Grzesiuk and Carroll, 1987). More recently, it was shown in Xenopus, silkworm, zebrafish, and mouse cells that a plasmid donor containing short (≤40 bp) regions of homology to a genomic target site can promote precise integration at the genomic cut site when the donor plasmid is cut adjacent to the homology (Nakade et al., 2014; Hisano et al., 2015; Aida et al., 2016). Gene targeting is likely mediated by the alternative-end joining/microhomology-mediated end joining (MMEJ) pathway or by a single-strand annealing (SSA) mechanism (Ceccaldi et al., 2016), collectively referred to as a homology mediated end joining (HMEJ). In contrast, in human cell culture, linear donors using a similar strategy with homologous ends have been reported to show inefficient integration until homology domains reach ~600 bp (Zhang et al., 2017), suggesting different repair pathways may predominate depending on cell type. In the initial reports using short regions of homology for in vivo gene targeting in zebrafish, the level of mosaicism in F0 injected animals was high, resulting in inefficient recovery of targeted alleles through the germline (Nakade et al., 2014; Hisano et al., 2015; Aida et al., 2016; Luo et al., 2018). Most recently, studies in Drosophila show efficient integration of exogenous DNA in flies and S2 cells using 100 bp homology arms flanked by a CRISPR target site for in vivo homology liberation (Kanca et al., 2019). Together, these studies suggest that a strategy that combines short homology flanked donors with in vivo homology arm liberation should lead to efficient precision targeting in zebrafish and mammalian cells.


Here, we present GeneWeld, a HMEJ strategy for targeted integration directed by short homology that we demonstrated leads to efficient germline transmission rates for recovery of targeted alleles in zebrafish (Wierson et al., 2020). We provide a suite of donor vectors, called pGTag, that can be easily engineered with homologous sequences (homology arms) to a gene of interest and a web interface for designing homology arms (Mann et al., 2019). Homology of 24 or 48 base pairs directly flanking the cargo DNA promotes efficient gene targeting in zebrafish, with germline transmission rates averaging approximately 50%. The tools and methodology described here provide a tractable solution to creating precise targeted integrations and open the door for more advanced genome editing strategies using short homology. The following detailed protocol outlines steps for gRNA selection, homology arm design, vector construction, CRISPR/Cas targeting, and recovery of targeted integration alleles.


Materials and Reagents

  1. Polystyrene Petri dishes (Thermo Fisher, catalog number: FB0875713)

  2. Borosilicate Glass Capillaries (World Precision Instruments, catalog number: 1B100-04)

  3. Microloader tips (Eppendorf, catalog number: 920001007)

  4. Commercially available molds for injection plates available from https://www.agnthos.se/index.php?id_product=204&controller=product

  5. Kwik-Fill borosilicate glass capillaries (World Precision Instruments, catalog number: 1B100-4)

  6. EasyStrip Plus Tube Strip with Attached Ultra Clear Caps (Thermo Fisher Scientific catalog number: AB2005)

  7. pGTag vectors are available through Addgene (https://www.addgene.org/kits/essner-geneweld/)

  8. NEB Stable Competent E. coli (New England Biolabs, catalog number: C3040I)

  9. One Shot TOP10 Chemically Competent E. coli (Thermo Fisher, Invitrogen, catalog number: C404010)

  10. pT3TS-nCas9n expression vector (Addgene, catalog number: 46757)

  11. PureYield Plasmid Miniprep System (Promega, catalog number: A1223)

  12. Ambion mMessage Machine T3 Transcription Kit (Thermo Fisher, catalog number: AM1348)

  13. miRNeasy Mini Kit (Qiagen, catalog number: 217004)

  14. NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, catalog number: E7645L)

  15. Zebrafish Tg (miniTol2<14XUAS:mRFP, γcry:GFP>)tl2 (Balciuniene et al., 2013)

  16. Zebrafish wild-type WIK strain (Zebrafish International Resource Center, catalog number: ZL84, https://zebrafish.org/home/guide.php)

  17. Agarose (Thermo Fisher, catalog number: BP160-500)

  18. Ethidium Bromide (Fisher Scientific, catalog number: BP1302-10)

  19. Ethyl 3-aminobenzoate methanesulfonate, Tricaine MS-22 C9H11NO2·CH4SO3 (Millipore Sigma catalog number 886-86-2)

  20. 1-phenyl-2-thiourea C7H8N2S (Thermo Fisher, catalog number: AC207250050)

  21. NorthernMax-Gly Sample Loading Dye (Thermo Fisher, catalog number: AM8551)

  22. Decon ElIMINase (Fisher Scientific, catalog number: 04-355-31)

  23. Molecular Grade RNase/DNase-Free water (e.g., Invitrogen, catalog number: 10977023)

  24. XbaI restriction endonuclease (New England Biolabs, catalog number: R0145S)

  25. BfuAI restriction endonuclease (New England Biolabs, catalog number: R0701S)

  26. BspQI restriction endonuclease (New England Biolabs, catalog number: R0712S)

  27. GoTag Green 2× MasterMix (Promega, catalog number: M7123)

  28. X-Gal solution, ready-to-use, 20mg/ml (Thermo Fisher Scientific, catalog number: R0941)

  29. LB broth (Fisher Scientific, catalog number: BP9723-500)

  30. LB agar (Fisher Scientific, catalog number: BP9724-2)

  31. SOC medium (Thermo Fisher Scientific, catalog number: 15544034)

  32. Kanamycin (Fisher Scientific, catalog number: BP9065)

  33. T4 Quick Ligase, Rapid DNA Ligation Kit (Thermo Fisher Scientific, catalog number: K1422)

  34. Sodium Hydroxide NaOH (Millipore, Sigma-Aldrich, catalog number: 30620)

  35. Tris Base (Fisher Scientific, catalog number: BP152-500)

  36. UgRNA

    5’-GGGAGGCGUUCGGGCCACAGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGGAUC-3’ and gene-specific sgRNAs ordered from Synthego (https://orders.synthego.com/products/crisprevolution-sgrna-ez-kit-13/#/tubes?mod_code=1) or IDT https://www.idtdna.com/site/order/oligoentry/index/crispr (see Section B.)

  37. Primers, can be ordered from IDT:

    F3'-check: 5'-GGCGTTGTCTAGCAAGGAAG-3'

    R3’ pgtag seq: 5'-ATGGCTCATAACACCCCTTG-3'

    R-Gal4-5'juncM: 5’-GCCTTGATTCCACTTCTGTCA-3’

    R-RFP-5'junc: 5’-CcttaatcagttcctcgcccttagA-3’

    R-eGFP-5'-junc: 5’-gctgaacttgtggccgttt-3’

    F-Gal4-3'juncM: 5’-GCAAACGGCCTTAACTTTCC-3’

    F-Gal4-3'junc: 5’-CTACGGCGCTCTGGATATGT-3’

    F-RFP-3'junc: 5’-cgacctccctagcaaactgggg-3’

    F-eGFP-3'junc: 5’-Acatggtcctgctggagttc-3’

  38. Zebrafish embryo E2 Medium (see Recipes)

Equipment

  1. Microcap Microliter Pipets (Drummond Scientific, catalog number: 1-000-0010)

  2. Flaming/Brown Micropipette Puller (Sutter Instrument, model: P-97)

  3. X-Cite 120W Metal Halide lamp (Excilitas Technologies, model: X-Cite 120Q)

  4. Pico-Injector (Harvard Apparatus, model: PLI-100)

  5. MM-3 Micromanipulator (Narishige, model: MM-3)

  6. Nitrogen gas tank, or JUN-AIR Oil-lubricated Piston Air Compressor (Cole-Parmer, catalog number: 1152000)

  7. Nanodrop (Thermo Fisher Scientific, model: NanoDrop 2000)

  8. iBright FL1500 Imaging System (Thermo Fisher Scientific, model: A44241)

  9. Shaking Incubator (Thermo Scientific, model: MaxQ8000)

  10. Isotemp Standard Laboratory Incubator (e.g., Thermo Scientific, model: 51-028-065HPM)

  11. Precision General Purpose Baths (e.g., Thermo Scientific, model: TSGP02)

  12. Thermal Cycler (Eppendorf, 6335000020)

  13. Zeiss SteREO Discovery.V8 Stereomicroscope or similar and epi-illumination X-Cite 120W metal halide light source with fiber optic cable (Excilitas Technologies)

  14. Owl EasyCast B2 Mini Gel Electrophoresis Systems (Thermo Fisher Scientific, model: B2-12)

Software

  1. GTagHd, Gene Sculpt Suite www.genesculpt.org/gtaghd/ (Mann et al., 2019)

  2. CRISPRScan http://www.crisprscan.org/ (Moreno-Mateos et al., 2015)

  3. Primer 3 http://biotools.umassmed.edu/bioapps/primer3_www.cgi

  4. Synthego ICE Analysis https://ice.synthego.com/#/

  5. Cas-Analyzer at CRISPR RGEN Tools http://www.rgenome.net/cas-analyzer/#

  6. GraphPad Prism https://www.graphpad.com/scientific-software/prism/

Procedure

The GeneWeld protocol is associated with the publication Wierson et al. (2020).


Table of Contents

  1. Introduction

  2. Selection of a CRISPR/spCas9 sgRNA target site, ordering sgRNAs, and in vitro synthesis of spCas9 mRNA

  3. Injection of sgRNA and spCas9 mRNA

  4. Testing for sgRNA mutagenesis efficiency and indel production

  5. Design short homology arms for pGTag targeting vector assembly

  6. One Pot Cloning of Homology Arms into pGTag Vectors

  7. Injection of GeneWeld reagents (spCas9 mRNA, Universal sgRNA (UgRNA), genomic sgRNA, and pGTag short homology vector) into 1-cell zebrafish embryos

  8. Test injected embryos for evidence of precision on-target integration

  9. Establish a new transgenic line of a precision targeted integration allele


  1. Introduction

    The GeneWeld strategy (Figure 1) and pGTag vector series are designed for straightforward assembly of vectors containing short homology arms for efficient CRISPR/Cas9-directed recovery of germline precision targeted integration alleles.



    Figure 1. Targeted integration of pGTag vector cargo DNA into a 5’ coding exon. Short homology arms complementary to the 5’ (green) and 3’ (blue) sequences of the genomic target site are cloned on the 5’ and 3’ sides of the vector cargo DNA. The short homology arm cargo cassette is flanked by two universal guide RNA UgRNA sites. CRISPR/Cas9 simultaneously targets double-strand breaks at the sgRNA genomic target site and at the UgRNA sites flanking the cargo on the plasmid donor. Exonuclease end resection liberates single-stranded DNA in the vector homology arms that is complementary to the resected strands on the 5’ and 3’ sides of the genomic double-strand break. The complementary sequences direct homology mediated end joining integration of the cargo DNA at the exon target site. PAM sequences are underlined, and small red arrows indicate Cas9 cut sites in the genome and vector.


  2. Selection of a CRISPR/spCas9 target site in the gene of interest

    1. Zebrafish wild-type strains in common use are polymorphic.

      Note: It is highly recommended to first sequence the target exon in the genomic DNA from your fish strain and use this sequence to design gRNAs.

    2. To identify an sgRNA site for targeting, first, view the gene model on a genome browser and download the gene sequences.

      1. At <ensembl.org> Search for the gene name of interest for the species of interest and open the Transcript page (Figure 2).



        Figure 2. Screenshot of zebrafish hand2 Transcript page on the ensembl.org genome browser (https://useast.ensembl.org/Danio_rerio/Transcript/Summary?db=core;g=ENSDARG00000008305;r=1:38193147-38195012;t=ENSDART00000020409)


      2. In the left-hand side bar click on “Exons” to find the first coding exon and initiation ATG (Figure 3).

        Note: If there are alternative transcripts for the gene, make sure there are not alternative initiation ATGs. If there are alternative start codons, target the first exon that is conserved in all transcripts to generate a strong loss-of-function allele.



        Figure 3. Exon sequences and Download page for zebrafish hand2 gene on the ensembl.org genome browser


      3. Click on the Download sequence button (Figure 3, red arrow). A list of choices for genomic, coding, untranslated regions, and intronic sequences is shown. Select the cDNA and target exon and download as separate sequence files.

      4. Using ApE <http://biologylabs.utah.edu/jorgensen/wayned/ape/> or SnapGene, annotate the coding sequence with the exons.

      5. Design primers to amplify the target exon from fin clip genomic DNA and sequence the amplicon. Use this sequence to identify gRNA sites either manually, by searching for PAM sequences, or using CRISPRScan. Remove additional “Gs” CRISPRScan added to the 5’ end. Select a sgRNA location that does not have an in-frame ATG downstream of the sgRNA target site. Annotate the selected target sequence and NGG PAM in the cDNA sequence files.

    3. Synthetic sgRNAs can be ordered from Synthego or IDT. On the Synthego ordering page, select “2’-O-Methyl at 3 first and last bases, 3’ phosphorothioate bonds between first 3 and last 2 bases” the Modifications tab.

    4. Design ~20 bp DNA primers for a PCR amplicon of ~130 bp of DNA surrounding the sgRNA target site. These primers will be used to amplify genomic DNA from embryos after injection of CRISPR reagents to test for mutagenesis at the target site. The presence of indels at the target site can be detected in the PCR products in multiple ways, including gel electrophoresis to visualize heteroduplex formation, resistance to restriction enzyme digestion at a site overlapping the sgRNA target and direct sequencing followed by ICE Analysis.

      1. Users can design primers with Primer 3 http://biotools.umassmed.edu/bioapps/primer3_www.cgi.

      2. Paste DNA sequence surrounding the target site into the web interface. It is recommended to use 160-300 bp of exon sequence centered on the cut site for primer design. Intron sequence can be used, but this often contains polymorphisms that can lead to amplification failure.

      3. Locate the target sequence, including the PAM sequence NGG (underlined, Bold in the example below), and predict the cut site (3 bp upstream of the PAM represented here by the ‘x’). Mark the targeted exon sequence approximately 65-150 bp on both sides of the cut site by putting [square brackets, highlighted in yellow] around it. Primer3 will design primers outside this sequence. This design allows the primers to be used for both checking of mutagenesis and for junction fragment analysis when checking for integration.


        Example:
        CGGCCTCGGGATCCACCGGCC[AGAATCGATATACTACGATGAACAGAGCAAATTTGTGTGTAATACGGTCGCCACCATGGCCTxCCTCGGTTTGCTACGATGCATTTGCACCACTCTCTCATGTCCGGTTCTGGG]AGGACGTCATCAAGGAGTTCATGCGCTTCAAGGTGCGCATGGAGGGCTCCGTGAAC


      4. Set the “Primer Size” variables to Min = 130, Opt = 170, and Max = 300. Everything else can be left at the defaults.

      5. Click on “Pick Primers.”

      6. Select primers from the output. Note the “product size” expected and the “tm” or melting temperature of each primer/pair. Mutagenesis is easier to visualize in smaller product sizes.


    5. Preparation of SpCas9 mRNA

      1. Digest ~5-10 μg pT3TS-nCas9n plasmid (plasmid Addgene #46757) (Jao et al., 2013) with Xba1 to linearize the vector.

      2. Purify linearized DNA with a Qiagen PCR cleanup kit or Promega PureYield Plasmid Miniprep System. Elute in RNase-free water.

      3. Run 100-500 ng on a 1.2% agarose gel in 1× TAE to confirm the plasmid is linearized.

      4. Use 100 ng to 1 μg DNA as template for in vitro transcription reaction with the mMESSAGE mMACHINE T3 kit Life Technologies (AM1348). Follow the manufacturer’s instructions provided with the kit. Save a 1 ul aliquot of the in vitro synthesis reaction.

      5. Use the miRNeasy Qiagen kit the purify the nCas9n mRNA according to the manufacturer’s instructions.

      6. Verify mRNA integrity by running a sample of the in vitro synthesized mRNA on a gel, before and after the miRNeasy Qiagen kit cleanup. Mix 1 μl of Cas9 mRNA, 4 μl of Molecular Grade RNase/DNase-free water, and 5 μl glyoxal dye (NorthernMax-Gly Sample Loading Dye, Thermo Fisher, AM8551).

      7. Heat mixture at 50 °C for 30 min in a water bath or thermal cycler, then place on ice.

      8. Clean the gel box, comb, and tray with Decon ElIMINase and rinse with DI water.

      9. Run all 10 μl of RNA mixture on a 1.2% agarose gel in 1× TAE at 100 V for 1 h as described above. Image gel in an iBright FL1500 Imaging System or another gel-documentation imaging system. One band should be visible at ~4.5 kb.

      10. Determine the concentration of the RNA sample using a Nanodrop. Concentrations between 0.45 and 1 μg/μl are expected.

      11. Aliquot and store RNA at -80 °C.


  3. Injection of sgRNA and spCas9 mRNA

    Deliver 25 pg sgRNA and 300 pg Cas9 mRNA in a 2 nl volume to embryos at the one-cell stage. Below is a step-by-step protocol for zebrafish embryo injection.

    1. A detailed video of zebrafish embryo injection can be found in Rosen et al. (2009). Cast zebrafish embryo injection trays with custom molds that create 45° troughs for lining up and holding embryos (Figure 4A). Molds are also available commercially (see Materials and Reagents).

      Melt 1.2% agarose in 1× E2 Medium and pour into a polystyrene Petri dish. The mold is set on top (Figure 4B), and, once the plates have set, gently remove the mold (Figure 4C). Injection trays can be used multiple times and are stored inverted at 4 °C for up to three weeks between use.



      Figure 4.Injection tray mold. The injection tray mold (A) is set on top of melted 1.2% agarose (B). Solidified injection plate with troughs to hold embryos (C).


    2. Pre-warm injection trays to 28.5°C prior to injection by placing them in a 28.5°C incubator for 20 min.

    3. Pull microcapillary glass needles using Kwik-Fill borosilicate glass capillaries on a Sutter Instrument Flaming/Brown Micropipette Puller.

    4. Prepare injection samples containing the following diluted in Molecular Grade RNase/DNase-free water:

      12.5 ng/μl of genomic sgRNA

      150 ng/μl of mRNA for Cas9

      Keep injection solution on ice

    5. Backload needles with injection solution using microloader pipet tips and attach to a micromanipulator. Connect the needle holder tubing to a Harvard Apparatus PLI-90 Pico-injector. Turn on nitrogen gas or the air compressor to pressurize the system and set injection pressure to 40 PSI with an injection time of 100-200 ms.

    6. Calibrate injection needle by first breaking the end of the tip off with sterile tweezers (Figure 5A, B). Use the pedal to expel 10 droplets and capture each droplet with a 30 mm long capillary tube that represents a volume of 1 μl (Figure 5C). Measure the distance from the end of the capillary to the meniscus of the liquid and convert to a volume of 1 mm = 30 nl; therefore, 2/3 of a mm = 20 nl. The volume of each droplet is adjusted by changing the injection time to achieve 2 nl droplets. There is a linear relationship between volume and time at a set pressure. Avoid injection times lower than 100 ms and higher than 400 ms.

    7. Transfer one-cell embryos from collection Petri dishes to the troughs on the pre-warmed agarose injection tray (Figure 5D). Each embryo is encased in a chorion. As the embryos near the first cell division at 45 minutes after fertilization, the single cell is clearly visible atop the yolk (Figure 5D).

    8. Use the micromanipulator to pierce the needle through the chorion and into the embryo. Inject 2 nl of sample at the center of the yolk interface/boundary between the single cell and the yolk (Figure 5E, white arrow points to interface where needle tip is placed). Inject embryos before the first cell division begins.



      Figure 5. Microinjection needle calibration and zebrafish single-cell embryo microinjection. (A) Backloaded injection needle with closed tip. (B) A small portion at the tip of the needle is removed using forceps to create an open end. (C) A single droplet of injection solution is expelled by pressing on the injection apparatus pedal. The tip of a 1 μl Drummond capillary tube that was used to capture 10 drops is shown. (D) Embryos lined up in an injection tray trough with labels indicating the chorion, yolk, and single-cell embryo. (E) Needle inserted through the chorion and into the embryo. The tip of the injection needle is positioned at the yolk interface (white arrow) between the single cell on top and the yolk below. The image in (E) was published in Almeida et al. (2021).


    9. After injection, wash the embryos from the injection tray into a clean Petri dish with embryo media.

    10. Keep 20-40 embryos separate to use as uninjected controls.

    11. At 3-5 h post injection, remove any unfertilized or dead embryos from the dishes.


  4. Test for sgRNA mutagenesis efficiency and indel production

    1. Biallelic inactivation can lead to loss of function of essential genes that may be lethal. After injection, count and remove dead embryos from the dish. If all embryos are defective and unlikely to survive, reduce the amount of guide sgRNA that is injected to 12.5 pg. If embryos still fail to survive, reduce the amount of sgRNA further to 6.25 pg. As we reported previously, for a ubiquitously expressed, essential gene such as the tumor suppressor rb1, the amount of injected sgRNA needs to be reduced to 6.25 pg to recover viable juvenile fish that survive to adulthood and transmit germline gene-edited alleles (Solin et al., 2015).

    2. Digestion of embryos for isolation of genomic DNA for mutagenesis analysis.

      Extract genomic DNA from either individual or pools of five embryos from the same injection, aged between 1 and 5 dpf, using the following protocol previously published (Wierson et al., 2020).

      1. Dechorionate embryos if they have not emerged from the chorion.

      2. Place embryo into a PCR tube and remove as much of the fish water as possible. Collect at least three injected embryos and one uninjected control embryo.

      3. Add 20 μl of 50 mM NaOH per embryo.

      4. Heat the embryos at 95°C in a thermocycler for 30 min.

      5. Vortex samples and spin the tubes down. The embryos should be completely dissolved.

      6. Neutralize the samples by adding 1 μl of 1 M Tris pH 8.0 per 10 μl NaOH. Mix by vortexing, then spin down.

      7. Genomic DNA is stored at -20°C.

    3. Analysis of CRISPR/Cas9 mutagenesis efficiency at targeted gene locus.

      1. Remove genomic DNA samples from -20°C and place on ice to thaw. Keep thawed genomic DNA on ice at all times.

      2. Set up the following PCR reactions for each tube of embryo digested genomic DNA using the gene-specific Forward and Reverse primers that were designed to create an amplicon around the CRISPR/Cas9 target site.

        12.5 μl of 2× GoTaq Mastermix

        1 μl of Forward Primer (10 μM)

        1 μl of Reverse Primer (10 μM)

        1 μl of gDNA template (digested embryos)

        9.5 μl of nuclease-free water

        For a 25 μl total

      3. Flick the tubes to mix and briefly spin down the PCR reactions.

      4. Run the following PCR program to amplify the targeted locus.

        95°C 2 min

        95°C 30 s

        55°C 30 s 35 cycles

        72°C 30 s

        72°C 5 min

        4°C hold

      5. Run up to 7 μl of PCR product on a 2.5 to 3% agarose gel in 1× TAE, for 1 h at 80-100V. Image gel in the iBright FL1500 Imaging System or another gel-documentation imaging system.

        An example of sgRNA injection and validation targeted exon 1 of the hand2 gene is shown in Figure 6A. The control uninjected embryo PCR amplicon runs as a single, tight band on the gel (Figure 6B, U). The amplicons from eight injected embryos show multiple bands or are diffuse in appearance relative to the control (Figure 6B, 1-8). This indicates heteroduplex formation in the PCR product caused by the presence of indel mutations at the CRISPR target site in the gene of interest.



        Figure 6. hand2 exon 1 sgRNA validation. A. Sequence of the hand2 reverse strand sgRNA site located in exon 1. B. PCR amplicons with primers flanking the sgRNA target site. Diffuse bands in injected embryos represent heteroduplex DNA caused by indel mutations at the target site.


      6. For quantitative analysis of mutagenesis efficiency, Sanger sequence PCR products to verify the presence of indels. The percentage of indel formation can be analyzed using Synthego’s ICE Analysis. Alternatively, Illumina MiSeq multiplex next-generation sequencing can be used to test the efficiency of multiple gRNAs in parallel.

        An example of ICE analysis of amplicons from the hand2 exon 1 targeted embryos #3 and #6 from Figure 6 is shown below in Figure 7. Embryo #3 and #6 show 84% and 80% indel alleles after targeting, respectively, indicating high mutagenesis efficiency of the sgRNA.



        Figure 7. Validation of sgRNA mutagenesis efficiency by ICE analysis. PCR amplicons from hand2 exon 1 targeted embryos #3 (A) and #6 (B) were Sanger sequenced and the results analyzed with Synthego ICE Analysis, revealing 84% and 80% of sequences contained indel mutations. Plots on the right show the range of indel mutations recovered.


  5. Design short homology arms for pGTag targeting vector assembly

    Homology-directed gene targeting allows the seamless integration of exogenous DNA into the genome with precise repair events at the target site. However, designing and cloning individual targeting vectors and homology arms for each gene of interest can be time consuming. The pGTag vector series and web design tools provide versatility and ease to generate knock-out alleles (Figure 8). The vectors contain BfuAI and BspQI type II restriction enzymes for cloning of short homology arms (24 or 48 bp) using Golden Gate cloning. The pGTag vectors require in-frame integration for proper reporter gene function. The reporter gene consists of several parts. A 2A peptide sequence causes translational skipping, allowing the following protein to dissociate from the locus peptide. The eGFP, TagRFP, or Gal4VP16 reporter coding sequences have several options for localization signals, including cytosolic (no signal), a nuclear localization signal (NLS), or a membrane localization CAAX sequence. Finally, translation is terminated by one of two different transcription termination polyadenylation (pA) sequences, the 3’UTR region of the zebrafish -actin gene or the SV40 viral transcription termination sequence.

    For many genes, the level of endogenous gene expression is not high enough to produce a detectable fluorescence signal from the integrated reporter gene. The Gal4VP16 pGTag vector in combination with the transgenic Tol2<14XUAS/RFP> reporter line (Balciuniene et al., 2013) allows for amplification of the signal. The Tol2<14XUAS/RFP> reporter line is available upon request from the lab of Dr. Darius Balciunas, Temple University.

    Plasmid sequence maps can be downloaded at www.genesculpt.org/gtaghd/.



    Figure 8. The pGTag vectors allow one step cloning of homology arms.


    All vectors (Figure 8) can be obtained through Addgene (www.addgene.org). Because the pGTag plasmids contain repeated sequences, vector recombination can occur in bacteria. We recommend using NEB Stable Competent E. coli (New England Biolabs, C3040I). Bacteria should be grown at 30°C to further reduce vector recombination.

    Homology Arm Design at GTagHD

    The web tool GTagHD allows for quick design of oligos to generate short 24 or 48 bp homology arms complementary to the target site in a gene of interest.

    Two complementary oligos with overhangs are annealed to generate the double stranded homology arm for cloning into the pGTag vector.

    To use the tool, choose the "Submit Single Job" tab. Follow the instructions in the tab.

    The sequences of two pairs of complementary oligos will be returned, one pair for the 5’ homology arm and the other for the 3’ homology arm. If there are problems with the sequences and values that were entered, the web page will display the errors and advice on how to fix them. Double-check your output as below.

    Manual Homology Arm Design

    The following protocol describes how to design homology arm oligos manually:

    Note: In the following section, orientation of target sites and homology is in the context of the reading frame of the genetic locus of interest. Example: A forward strand CRISPR gRNA means that the gRNA and PAM are encoded on the sense strand of the gene. Upstream homology domains are 5’ to the CRISPR/Cas9 cut site, and downstream homology domains are 3’ to the cut site with respect to the reading frame of the gene being targeted.

    Also note: Upper case and lower case bases are not specially modified; this is simply a visual marker of the different parts of the homology arms.


    Upstream Homology Arm Design

    1. Open the sequence file for the gene of interest and identify the CRISPR site (in this example, it is a Reverse CRISPR target in Yellow, the PAM is in Orange, and the coding sequence is in purple). Copy the 48 bp 5’ of the CRISPR cut (the highlighted section below) into a new sequence file; this is the upstream homology (Figure 9).



      Figure 9. Screenshot of a targeted gene displayed in ApE, highlighting the target sequence (yellow), PAM (orange), coding sequence (purple), and the gene sequence of the upstream homology arm (highlighted white).


    2. Observe the next three bases immediately upstream of the 48 bp of homology, and pick a base not present to be the 3 bp spacer between the homology and the Universal PAM in the vector. Here, the three bases are “GGA,” so “ccc” was chosen for the spacer. Add the spacer to the new file 5’ (in front) of the homology (see below). The spacer acts as a non-homologous buffer between the homology and the eventual 6 bp flap from the universal guide sequence that will occur when the cassette is liberated and may improve intended integration rates over MMEJ events (Figure 10).



      Figure 10. Screenshot of the gene sequence of the upstream homology arm (purple), the PAM (orange), and the remaining target sequence to the cut site (yellow). ccc was added as a spacer with a non-homologous sequence.


    3. Determine where the last codon is in the homology. Here, the 3’ G in the homology domain is the first base in the codon cut by this CRISPR target. Complete the codon by adding the remaining bases (called padding on GTagHD) for that codon from your sequence to ensure your integration event will be in frame (Figure 11).



      Figure 11. Screenshot of the gene sequence of the upstream homology arm (purple), the PAM (orange), and the remaining target sequence to the cut site with the padding nucleotides (tg) to keep the integration in frame (yellow).


    4. Add the BfuAI enzyme overhang sequences for cloning to the ends of the homology domain. Here, both overhangs, 5’-GCGG and 3’-GGAT, are added to prevent errors in copying sequence for the oligos in the next two steps (Figure 12).



      Figure 12. Screenshot of the gene sequence of the upstream homology arm (purple), the PAM (orange), and the remaining target sequence to the cut site with the padding nucleotides (tg) to keep the integration in frame (yellow) and the BfuAI sites added to each end.


    5. The Upstream Homology Oligo A will be this sequence from the beginning to the end of the last codon (see highlighted below). Copy and paste this sequence into a new file and save it. In this example, this oligo sequence is 5’-GCGGcccGTTTTCTTACGCGGTTGTTGGATGAAATCTCCAACCACTCCACCTTCGtg-3’ (Figure 13).



      Figure 13. Screenshot of the gene sequence of the upstream homology arm (purple), the PAM (orange), and the remaining target sequence to the cut site with the padding nucleotides (tg) to keep the integration in frame (yellow) and the BfuAI sites added to each end. The sequence of Oligo A is in white.


    6. The Upstream Homology Oligo B will be the reverse complement of this sequence from the beginning of the spacer to the end of the sequence (see highlighted below in Figure 14). Copy the reverse complement, paste it into a new file, and save it. In this example, this oligo sequence is 5’-ATCCcaCGAAGGTGGAGTGGTTGGAGATTTCATCCAACAACCGCGTAAGAAAACggg-3’.



      Figure 14. Screenshot of the gene sequence of the upstream homology arm (purple), the PAM (orange), and the remaining target sequence to the cut site with the padding nucleotides (tg) to keep the integration in frame (yellow) and the BfuAI sites added to each end. The sequence of Oligo B is highlighted. Use the reverse complement of the highlighted sequence.


    Downstream Homology Arm Design

    1. Open sequence file for the gene of interest and identify the CRISPR site. The reverse CRISPR target is in Yellow, the PAM in Orange, and coding sequence in purple. Copy the 48 bp 3’ of the CRISPR cut into a new sequence file; this is the downstream homology (Figure 15).



      Figure 15. Screenshot of a targeted gene, highlighting the target sequence (yellow), PAM (orange), coding sequence (purple), and the gene sequence of the downstream homology arm (in white).


    2. Observe the next three bases downstream of the 48 bp of homology and pick a base not present to be the 3 bp spacer between the homology and the Universal PAM in the vector. Here, the bases are “CTG;” therefore, “aaa” was chosen for the spacer. Add the spacer to the new file 3’ of (after) the homology (Figure 16).



      Figure 16. Screenshot of the gene sequence in the downstream homology arm from the targeted gene. This comprises part of the target sequence (yellow) and additional 3’ coding sequence (purple). aaa was added as padding nucleotides.


    3. Add the BspQI enzyme overhang sequences for cloning to the ends of the homology domain. Here, both overhangs, 5’-AAG and 3’-CCG, are added to prevent errors in copying sequence for the oligos in the next two steps (Figure 17).



      Figure 17. Screenshot of the gene sequence in the downstream homology arm from the targeted gene with part of the target sequence (yellow) and additional 3’ coding sequence (purple). BspQI enzyme overhang sequences are added to each end.


    4. The Downstream Homology Oligo A will be this sequence from the beginning of the sequence to the end of the spacer (see highlighted below). In this example, this oligo sequence is 5’-AAGTGGGCAAGATATGGCTCACGTTATTCATCATCTTCCGCATTGTTTTGAaaa-3’ (Figure 18).



      Figure 18. Screenshot of the gene sequence in the downstream homology arm from the targeted gene with part of the target sequence (yellow) and additional 3’ coding sequence (purple). The sequence for Oligo A is in white.


    5. The Downstream Homology Oligo B (will be the reverse complement of this sequence from the beginning of the homology to the end of the sequence (see highlighted below). In this example this oligo sequence is 5’-CGGtttTCAAAACAATGCGGAAGATGATGAATAACGTGAGCCATATCTTGCCCA-3’ (Figure 19).



      Figure 19. Screenshot of the gene sequence in the downstream homology arm from the targeted gene with part of the target sequence (yellow) and additional 3’ coding sequence (purple). The sequence for Oligo B is highlighted in white. The reverse complement should be ordered.


    6. An example of correct homology arm design, showing complementary overhangs for cloning into the pGTag and pPRISM BfuAI and BspQ1 sites for a hand2 gRNA site (Figure 20).



      Figure 20. Example of pGTag and pPRISM vector homology arm design showing complementary 5’ overhangs for cloning into the BfuAI and BspQI type II restriction enzyme sites. Diagram of CRISPR/Cas9 target site in the hand2 gene. gRNA sequence in red and PAM sequence underlined and in bold. Annealed homology arm oligos A and B are shown with overhangs (green) complementary to the vector overhangs after enzyme digestion. n, spacer nucleotides; n., nucleotides included to maintain the reading frame of pGTag integration alleles.


  6. One Pot Cloning of Homology Arms into pGTag Vectors

    Notes:

    1. If the homology arm oligos contain the 5’-ACCTGC-3’ or 5’-GAAGAGC-3’ sequences (or their complements), the cloning reaction will be less efficient.

    2. If One Plot cloning is unsuccessful, the 5’ and 3’ homology arms can be cloned sequentially into the vector using gel-purified linear plasmids digested with the appropriate enzyme.

    1. Homology Arm Annealing

      Anneal upstream and downstream homology oligo pairs separately:

      4.5 μl oligo A at 10 μM

      4.5 μl olio B at 10 μM

      4 μl 10× Buffer 3.1 from NEB

      27 μl dH2O

      For a 40 μl total

      To anneal the oligos, run the following program in a thermal cycler: Step 1, incubate at 98°C for 5 minutes; Step 2, incubate at 97°C for 45 seconds; repeat Step 2 for 90 cycles during which the temperature is decreased by 1°C/cycle; hold at 4°C for the final step. Alternatively, boil water in a glass beaker on a hot plate and incubate the tube in the boiling water for 5 minutes. Remove the beaker from the heat source and allow it to cool to room temperature. Store the annealed homology arms on ice or in a -20°C freezer.

    2. 1-Pot Digest

      Mix the following solutions:

      4.0 μl dH2O

      2 μl Plasmid at 50 ng/μl

      1 μl 10× Buffer 3.1 from NEB

      1 μl 5' annealed homology arm

      1 μl 3' annealed homology arm

      0.5 μl BfuAI enzyme from NEB

      0.5 μl BspQI enzyme from NEB

      For a10 μl total

      Incubate at 50°C for 1 h, place on ice.

    3. Ligation

      Add the following:

      3 μl 5× T4 quick ligase buffer

      1.5 μl dH2O

      0.5 μl T4 quick ligase

      For a 15 μl total

      Incubate 8-10 min at room temperature (to overnight). Store at -20°C.

    4. Transformation: To prevent recombination at repetitive elements in the plasmid, grow transformations and overnight cultures at 30°C. Our standard protocol uses NEB Stable Competent E. coli (C3040H) cells for cloning and propagation of the GeneWeld pGTag and pPRISM plasmid series to limit recombination.

      1. On ice, thaw 1 (one) vial competent cells (50 μl) for every 2 ligation reactions (approx. 5 min).

      2. While cells are thawing, label the microcentrifuge tubes for each ligation and put on ice.

      3. Once the cells have thawed, use a pipette to transfer 25 μl of the competent cells into each labeled tube.

      4. Add 1.5 μl of a ligation reaction into competent cells to transform.

        The amount of ligation reaction added should be less than 5% of the volume of competent cells.

      5. Mix by tapping the tube several times or gently mixing with the pipet tip.

        Note: Do NOT mix by pipetting; this will lyse the cells.

      6. Incubate on ice for 5 to 20 min.

      7. Heat-shock the cells by submerging the portion of the tube containing the cells in a 42°C water bath for 40-50 s.

      8. Incubate cells on ice for 2 min.

      9. Add 125 μl of room temperature LB or SOC to each transformation.

      10. Incubate cells at 30°C for 1-1.5 h in a shaking incubator.

      11. While the transformed cells are recovering, spread 40 μl of X-Gal solution and 40 μl IPTG 0.8 M on LB Kanamycin selection plates.

      12. X-Gal is lethal to cells while wet; it is recommended to first label the plates and then place them in a 30°C incubator to dry.

      13. After recovery and the X-Gal is dry, plate 150 μl of each transformation on the corresponding correctly labeled plate.

      14. Incubate plates overnight at 30°C.

    5. Growing colonies

      Pick three white colonies from each plate and grow in separate glass culture tubes with 3 ml LB/Kanamycin, overnight at 30°C, or to pre-screen colonies by colony PCR:

      1. Pick up to 8 colonies with a pipet tip and resuspend them in separate aliquots of 5 μl dH2O. Place the tip in 3 ml of LB/Kan, label, and store at 4°C.

      2. Make a master mix for your PCR reactions containing the following amounts times the number of colonies you picked.

        7.5 μl 2× GoTaq mastermix

        5.5 μl dH2O

        0.5 μl primer at 10 μM “F3'-check” 5'-GGCGTTGTCTAGCAAGGAAG-3'

        0.5 μl primer at 10 μM “3’_pgtag_seq”5'-ATGGCTCATAACACCCCTTG-3'

        For a 14 μl total

      3. Aliquot 14 μl of mixed master mix into separate labeled PCR tubes.

      4. Add 1 μl of colony to each reaction as template, or 20 ng purified plasmid as control.

      5. Cycle in a thermocycler

        95°C 2 min

        95°C 30 s

        57°C 30 s 35 cycles

        72°C 30 s

        72°C 5 min

        4°C hold

      6. Run 5 μl of PCR product on a 1% agarose gel. Image gel on iBright FL1500 Imaging System or another gel-documentation imaging system. There should be bands that are a different size than the control.

    6. Mini Prep Cultures

      Follow Qiagen Protocol

    7. Sequencing of Plasmids

      The 5’ homology arm can be sequenced by the 5'_pgtag_seq primer:

      5'-GCATGGATGTTTTCCCAGTC-3’.

      The 3’ homology arm can be sequenced with the “3’_pgtag_seq”primer:

      5'-ATGGCTCATAACACCCCTTG-3'.


  7. Injection of GeneWeld Reagents (spCas9 mRNA, Universal sgRNA (UgRNA), genomic sgRNA, and pGTag homology vector) into 1-cell zebrafish embryos

    Prepare and collect the following reagents for injection:

    1. Prepare nCas9n mRNA from pT3TS-nCas9n (Addgene #46757) (Jao et al., 2013) (See Section B 5. Preparation of SpCas9 mRNA).

    2. The UgRNA and genomic sgRNA can be directly ordered from IDT or Synthego and resuspended in Molecular Grade RNase/DNase-free water.

    3. The pGTag homology vectors should be purified a second time prior to microinjection under RNase free conditions with the Promega PureYield Plasmid Miniprep System beginning at the endotoxin removal wash. Plasmid DNA is eluted in Molecular Grade RNase/DNase-Free water.

    4. Embryo Injections for Integration of pGTag vectors.

      Injections are performed into single cell embryos at a volume of 2 nl per embryo containing the following concentration of RNAs and vector:

      In injection solution                     In embryo

      75 pg/nl of nCas9n mRNA    150 pg of nCas9n mRNA   

      12.5 pg/nl of genomic sgRNA     25 pg of genomic sgRNA   

      12.5 pg/nl of UgRNA             25 pg of UgRNA    

      5 pg/nl of pGTag DNA             10 pg of pGTag DNA   


  8. Test injected embryos for evidence of precision on-target integration

    1. Examine injected embryos for fluorescence under a Zeiss Discovery dissecting microscope with a 1× objective at 70-100× magnification. If weak signals are observed, manually dechorionate the embryos, and view on a glass depression well slide on a standard upright compound microscope with epi-illumination. High resolution confocal live imaging can also be carried out, as shown in Wierson et al. (2020) Figure 3 (https://elifesciences.org/articles/53968/figures#fig3).

      The type of light source used for fluorescent protein activation significantly affects the ability to visualize fluorescence signals. The X-Cite 120W metal halide light source with fiber optic cable (Excilitas Technologies) works well to visualize fluorescence after somatic targeting. The TagRFP protein also is shifted in its excitation, and filters optimized for this protein are recommended. Filters optimized for GFP and BFP are also recommended. If no or weak signal is observed, integration of pGTag-Gal4VP16 can be used to amplify reporter expression in the 14XUAS-RFP transgenic line (Balciuniene et al., 2013).

    2. Perform junction fragment PCR analysis on positive embryos that display widespread fluorescence in expression domains consistent with the targeted gene. Isolate genomic DNA from individual embryos and a control embryo (See Section D 2. Digestion of embryos for isolation of genomic DNA for mutagenesis analysis). PCR amplifies the genomic DNA-integrated cassette 5’ and 3’ junctions fragments. An example of F0 embryo junction analysis is shown in Figure 3s1 in Wierson et al., 2020 (https://elifesciences.org/articles/53968/figures#fig3s1).

      The following primers are used for junction fragment analysis and must be paired with gene-specific primers (5’ to 3’):

      5’ pGTag junctions:

      R-Gal4-5'juncM GCCTTGATTCCACTTCTGTCA and a gene-specific forward primer

      R-RFP-5'junc CcttaatcagttcctcgcccttagA

      R-eGFP-5'-junc gctgaacttgtggccgttt

      3’ pGTag junctions:

      F-Gal4-3'juncM GCAAACGGCCTTAACTTTCC and a gene specific reverse primer

      F-Gal4-3'junc CTACGGCGCTCTGGATATGT

      F-RFP-3'junc cgacctccctagcaaactgggg

      F-eGFP-3'junc Acatggtcctgctggagttc


      To control for PCR amplification artifacts as described in (Won and Dawid, 2017), perform PCR junction analysis on embryos injected with all targeting reagents minus the genomic sgRNA.

      The alternate primers F-Gal4-3'juncM and F-Gal4-3'juncJ may increase primer specificity, depending on the target gene.

      7.5 μl 2× GoTaq mastermix

      5.5 μl dH2O

      0.5 μl primer at 10 μM genomic primer

      0.5 μl primer at 10 μM pGTag primer

      For a 14 μl total

      1. Aliquot 14 μl of mixed master mix into separate labeled PCR tubes.

      2. Add 1 μl of genomic DNA to each reaction as template.

      3. Cycle in a thermocycler with the following steps:

      4. Run 5 μl of PCR product on a 1.2% agarose gel in 1× TAE. Image gel on the iBright FL1500 Imaging System or another gel-documentation imaging system. Putative junction fragments should give bands that are of the predicted size.


  9. Establish a new transgenic line of a precision targeted integration allele

    1. Raise to adulthood fluorescence reporter-expressing F0 siblings of injected embryos that showed positive bands on the 5’ and 3’ junction analysis, indicating precision targeted integration. Outcross F0 adults to wild type and examine the progeny for reporter gene fluorescence as described above to identify F1 embryos that have inherited a stable germline integration allele. For Gal4Vp16 integration alleles, cross the F0 adults to the 14XUAS:RFP reporter line. Silencing of the 14XUAS:RFP reporter may result in mosaic expression patterns in Gal4Vp16 targeted F1 embryos.

    2. Test F1 fluorescence positive embryos for precise transgene integration by junction fragment PCR analysis as described above. Raise F1 siblings to adulthood and fin-clip to identify individuals with precise targeted transgene integration as shown in Figure 4s 2-4 of Wierson et al. (2020) (https://elifesciences.org/articles/53968/figures#fig4s2).

    3. Outcross a single positive F1 adult to establish F2 families. F1s can also be sacrificed 3 weeks post-fertilization to the confirm location and precision of targeted integrations by genomic Southern Blot RFLP analysis as inFigure 4s1 of Wierson et al. (2020) (https://elifesciences.org/articles/53968/figures#fig4s1). Continue to maintain lines by outcrossing to wild type in subsequent generations. Tables 1 and 2 of Wierson et al. (2020) (https://elifesciences.org/articles/53968/figures) show the range of germline transmission frequencies of precision targeted integration alleles at eight zebrafish loci.

    4. To perform an initial assessment of whether the targeted integration allele causes a loss of function phenotype, F0 and F1 identified fish can be incrossed or crossed to a known indel allele of the targeted gene.

Data analysis

Links to numerical data in the original article (Wierson et al., 2020) are included in the protocol.

Recipes

Zebrafish embryo E2 Medium (Westerfield, 1995) (https://wahoo.cns.umass.edu/book/export/html/867)

Acknowledgments

This work was supported by the NIH grant R24OD020166 (JJE, MM, DD, KJC, and SCE). Research reported in this publication was made possible in part by the services of the Kansas University Genome Sequencing Core Laboratory supported by the National Institute of General Medical Sciences (NIGMS) of the NIH under award number P20GM103638. The GeneWeld protocol is associated with Wierson et al., 2020.

Competing interests

JJE, MM, and KJC have a financial conflict of interest with Recombinetics, Inc.; JJE and SCE with Immusoft, Inc.; JJE, MM, WAW, KJC, and SCE with LifEngine and LifEngine Animal Technologies.

Ethics

All zebrafish experiments described in this protocol were carried out under approved protocols from Iowa State University Animal Care and Use Committee Log#11-06-6252, in compliance with American Veterinary Medical Association and NIH guidelines for the humane use of animals in research.

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简介

[摘要]高效的精准基因组工程需要在基因组目标位点进行高频率和特异性的整合。先前已经报道了斑马鱼基因靶向的多种设计策略,用于整合等位基因的种系恢复频率差异很大。的GeneWeld协议和pGTag (p lasmids为ģ烯标签更改)向量系列提供一组资源的精简精度基因在斑马鱼中定位。我们的方法使用的24个短同源- 48 bp的推动有针对性的整合通过同源介导的末端连接(HMEJ)在CRISPR / DNA记者盒CAS大全MSDS诱导的DNA双-链断裂。该pGTag载体含有两侧记者通过通用CRISPR因组序列解放瞄准盒体内,揭露同源臂的同源性-驱动的集成。精密种系传输速率-定向整合等位基因范围22 - 100%。我们的系统提供一个精简的,直接的,和成本效益高的方式-效率基因打靶对斑马鱼的应用。

图文摘要:

用于 CRISPR/Cas9 靶向整合的GeneWeld方法。

[背景]设计师核酸酶迅速扩大,其中研究人员可以利用的方式Ë ndogenous DNA修复机制,用于创建基因敲除,记者基因敲除的插件,基因缺失,单核苷酸多态性,和表位-标记等位基因在不同的物种(比德尔等人,2012;Beumer 等人,2008 年;Carlson 等人,2012 年;Geurts 等人,2009 年;Yang 等人,2013 年)。基因组中的单个 dsDNA 断裂导致重组频率增加并促进基于同源重组 (HR) 的载体的整合(Hasty 等,1991;Hoshijima 等,2016;Orr-Weaver 等,1981;Rong和 Golic,2000 年;Shin 等人,2014 年;Zu 等人,2013 年)。此外,靶向载体的体外或体内线性化刺激同源定向修复 (HDR) (Hasty 等,1991;Hoshijima 等,2016;Jasin 和 Berg,1988;Orr-Weaver 等,1981;Rong和 Golic,2000 年;Shin 等人,2014 年;Zu 等人,2013 年)。在靶向双链断裂(DSB)利用HDR或HR允许定向敲入用碱外源DNA的-对精度; 然而,报告的频率差异很大,并且具有长同源臂的工程靶向载体并不简单。

以前的工作表明,非洲爪蟾卵母细胞能够连接或重组末端含有短同源区域的线性 DNA 分子,这种活性很可能由外切核酸酶活性介导,允许切除的同源性进行碱基配对(Grzesiuk 和 Carroll,1987 年) ) 。最近,在非洲爪蟾、家蚕、斑马鱼和小鼠细胞中表明,当供体质粒被切割时,含有与基因组靶位点同源的短(≤40 bp )区域的质粒供体可以促进基因组切割位点的精确整合。与同源性相邻(Aida 等人,2016 年;Hisano 等人,2015 年;Nakade 等人,2014 年)。基因靶向可能是由替代-末端接合/介导microhomology介导的末端连接(MMEJ)途径或通过单一-链退火(SSA)机制(Ceccaldi等人,2016) ,统称为一个同源性介导的末端连接(HMEJ) 。相比之下,在人类细胞培养中,据报道使用具有同源末端的类似策略的线性供体在同源结构域达到 ~600 bp之前表现出低效整合(Zhang 等,2017),表明不同的修复途径可能占主导地位,具体取决于细胞类型. 在使用短同源区域进行斑马鱼体内基因靶向的初步报告中,F0 注射动物的嵌合水平很高,导致靶向等位基因通过种系的恢复效率低下(Aida 等,2016;Hisano 等。 , 2015; Luo et al., 2018; Nakade et al., 2014) 。最近,对果蝇的研究表明,外源 DNA 在果蝇和 S2 细胞中有效整合,使用 100 bp同源臂,两侧是 CRISPR 靶位点,用于体内同源性释放(Kanca 等人,2019 年)。总之,这些研究表明,将短同源侧翼供体与体内同源臂解放相结合的策略应该可以在斑马鱼和哺乳动物细胞中实现有效的精确靶向。

在这里,我们提出了GeneWeld ,这是一种 HMEJ 策略,用于由短同源性指导的靶向整合,我们证明了该策略可提高斑马鱼靶向等位基因恢复的有效种系传输率(Wierson 等,2020)。我们提供了一套称为pGTag的供体载体,可以轻松地将其与感兴趣的基因的同源序列(同源臂)和用于设计同源臂的网络界面进行工程化(Mann 等人,2019 年)。ħ omology的24或48个碱基对直接侧翼的货物DNA促进有效的基因在斑马鱼靶向,与种系传递率平均为约50%。此处描述的工具和方法提供了一种易于处理的解决方案,可以创建精确的靶向整合,并为使用短同源性的更高级基因组编辑策略打开大门。以下详细方案概述了 gRNA 选择、同源臂设计、载体构建、CRISPR/ Cas靶向和靶向整合等位基因恢复的步骤。

关键字:CRISPR/Cas9, 基因敲入, 同源介导端连接, 靶向整合, 斑马鱼


材料和试剂

聚苯乙烯培养皿(Thermo Fisher,目录号:FB0875713)
硼硅酸盐玻璃毛细管(World Precision Instruments,目录号:1B100-04)
Microloader提示(Eppendorf,目录号:920001007)
市售注塑板模具可从https://www.agnthos.se/index.php?id_product=204&controller=product 获得
Kwik-Fill 硼硅酸盐玻璃毛细管(World Precision Instruments,目录号:1B100-4)
EasyStrip Plus Tube Strip 附有超透明帽(Thermo Fisher Scientific 目录号:AB2005)
pGTag载体可通过Addgene ( https://www.addgene.org/kits/essner-geneweld/ ) 获得
NEB 稳定的感受态大肠杆菌(New England Biolabs ,目录号:C3040I )             
One Shot TOP10 化学感受态大肠杆菌(Thermo Fisher ,Invitrogen,目录号:C404010 )                           
pT3TS-nCas9n 表达载体(Addgene ,目录号:46757 )
PureYield Plasmid Miniprep System (Promega ,目录号:A1223 )
Ambion mMessage Machine T3 转录套件(Thermo Fisher,目录号:AM1348 )
miRNeasy Mini Kit (Qiagen ,目录号:217004 )
NEBNext Ultra II DNA Library Prep Kit for Illumina (新英格兰生物实验室,目录号:E7645L )
斑马鱼Tg ( miniTol2<14XUAS:mRFP, γ cry:GFP > ) tl2 (Balciuniene et al., 2013)
斑马鱼野生-型WIK株(斑马鱼国际资源中心,目录号:ZL84,https://zebrafish.org/home/guide.php)
琼脂糖(Thermo Fisher,目录号:BP160-500 )
溴化乙锭(Fisher Scientific,目录号:BP1302-10 )
3-氨基苯甲酸乙酯甲磺酸乙酯,Tricaine MS-22 C 9 H 11 NO 2 ·CH 4 SO 3 (Millipore Sigma目录号886-86-2 )
1-苯基-2-硫脲C 7 H 8 N 2 S (Thermo Fisher,目录号:AC207250050 )
NorthernMax-Gly 上样染料(Thermo Fisher,目录号:AM8551 )
Decon ElIMINase (Fisher Scientific,目录号:04-355-31 )
分子级RNA酶/ DNA酶的水中(例如,Invitrogen公司,目录号:10977023 )
XbaI限制性核酸内切酶(New England Biolabs ,目录号:R0145S )
BfuAI限制性核酸内切酶(New England Biolabs ,目录号:R0701S )
BspQI限制性核酸内切酶(New England Biolabs ,目录号:R0712S )
GoTag Green 2 × MasterMix (Promega ,目录号:M7123 )
X-Gal 溶液,即用型,20mg/ml (Thermo Fisher Scientific,目录号:R0941 )
LB b罗斯(Fisher Scientific公司,目录号:BP9723-500 )
LB a gar (Fisher Scientific,目录号:BP9724-2 )
SOC米edium (热Fisher Scientific公司,目录号:15544034 )
卡那霉素(Fisher Scientific,目录号:BP9065 )
T4 Quick Ligase,Rapid DNA Ligation Kit (Thermo Fisher Scientific,目录号:K1422 )
氢氧化钠NaOH (Millipore ,Sigma - Aldrich,目录号:30620 )
Tris Base (Fisher Scientific,目录号:BP152-500 )
核糖核酸
5' - GGGAGGCGUUCGGGCCACAGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGGAUC - 3'和基因-特异性sgRNAs从有序Synthego (https://orders.synthego.com/products/crisprevolution-sgrna-ez-kit-13/#/tubes?mod_code=1)或IDT HTTPS: //www.idtdna.com/site/order/oligoentry/index/crispr (见B部分)
引物,可从 IDT 订购:
F3'-检查:5'-GGCGTTGTCTAGCAAGGAAG-3'
R3' pgtag seq : 5'-ATGGCTCATAACACCCCTTG-3'
R-Gal4-5'juncM : 5'- GCCTTGATTCCACTTCTGTCA-3'
R-RFP-5'jun c : 5'- CCTTAATCAGTTCCTCGCCCTTAGA-3'
R-eGFP-5'-jun c : 5'- GCTGAACTTGTGGCCGTTT-3'
F-Gal4-3'juncM : 5'- GCAAACGGCCTTAACTTTCC-3'
F-Gal4-3'junc : 5'- CTACGGCGCTCTGGATATGT-3'
F-RFP-3'junc : 5'- CGACCTCCCTAGCAAACTGGGG-3'
F-eGFP-3'jun c : 5'- ACATGGTCCTGCTGGAGTTC-3'
斑马鱼胚胎 E2 培养基(见食谱)

设备

Microcap Microliter Pipets(Drummond Scientific,目录号:1-000-0010)
火焰/棕色微量移液器拉拔器(萨特仪器,型号:P-97 )
X-Cite 120W 金属卤化物灯(Excilitas Technologies,型号:X-Cite 120Q )
Pico-Injector (哈佛仪器,型号:PLI-100 )
MM-3 显微操作器( Narishige ,型号: MM-3 )
氮气罐,或 JUN-AIR 油润滑活塞空气压缩机(Cole-Parmer,目录号:1152000 )
Nanodrop (赛默飞世尔科技,型号:NanoDrop 2000 )
iBright FL1500 成像系统(Thermo Fisher Scientific,型号:A44241 )
摇动培养箱(Thermo Scientific ,型号:MaxQ8000 )
ISOTEMP标准实验室孵化器(例如,热科学,型号:51-028-065HPM )
精密通用浴槽(例如,Thermo Scientific,型号:TSGP02 )
热循环仪(Eppendorf,6335000020 )
Zeiss SteREO Discovery.V8 立体显微镜或类似的和落射照明X-Cite 120W 金属卤化物光源,带光纤电缆(Excilitas Technologies)
Owl EasyCast B2 迷你凝胶电泳系统(Thermo Fisher Scientific,型号:B2-12 )

软件

GTagHd ,基因雕刻套件www.genesculpt.org/gtaghd/ (曼等人,2019 年)
CRISPRScan http://www.crisprscan.org/(莫雷诺马特奥斯等人。,2015 )
入门 3 http://biotools.umassmed.edu/bioapps/primer3_www.cgi
Synthego ICE 分析https://ice.synthego.com/#/
CRISPR RGEN 工具中的Cas- Analyzer http://www.rgenome.net/cas-analyzer/#
GraphPad棱镜https://www.graphpad.com/scientific-software/prism/

程序

所述GeneWeld协议与发布关联Wierson等。(2020 年)。

目录
介绍
一个CRISPR / spCas9的选择因组目标网站,订购sgRNAs ,并在体外合成spCas9 mRNA的
注射sgRNA和 spCas9 mRNA
测试的因组诱变效率和插入缺失生产
设计用于pGTag靶向载体组装的短同源臂
将同源臂一锅克隆到pGTag载体中
的注射GeneWeld试剂(spCas9的mRNA,环球因组(UgRNA ),基因组因组,并pGTag短同源性载体中)进入1细胞斑马鱼胚胎
测试注射的胚胎以证明精确的目标整合
建立新的精准靶向整合等位基因转基因品系

介绍
所述GeneWeld策略(图1)和pGTag向量系列被设计用于容纳短同源臂用于有效CRISPR / Cas9矢量的简单的装配-种系精度靶向整合等位基因的定向恢复。


图 1. pGTag载体货物 DNA靶向整合到 5' 编码外显子中。与基因组目标位点的 5'(绿色)和 3'(蓝色)序列互补的短同源臂克隆在载体货物 DNA 的 5' 和 3' 侧。短同源臂货物盒两侧是两个通用引导 RNA UgRNA位点。CRISPR / Cas9同时靶向双-在链断裂因组基因组目标站点和UgRNA点侧接质粒捐助货物。核酸外切酶切除端释放单-在所述载体同源臂链DNA也就是在5'和3'基因组双两侧被切除链互补-链断裂。互补序列指导货物 DNA 在外显子靶位点的同源介导的末端连接整合。PAM 序列带有下划线,红色小箭头表示基因组和载体中的 Cas9 切割位点。

在感兴趣的基因中选择 CRISPR/spCas9 靶位点
斑马鱼野生-通用型菌株是多态。
注意:强烈建议首先对来自您的鱼品系的基因组 DNA 中的目标外显子进行测序,然后使用该序列设计gRNA 。
要确定用于靶向的sgRNA位点,首先,在基因组浏览器上查看基因模型并下载基因序列。
搜索感兴趣物种的感兴趣基因名称并打开 Transcript 页面(图 2)。
图 2. ensembl.org 基因组浏览器中斑马鱼hand2 Transcript 页面的屏幕截图( https://useast.ensembl.org/Danio_rerio/Transcript/Summary?db=core;g=ENSDARG00000008305;r=1:38193147-38195012;tt =ENSDART00000020409 )

在左侧栏中单击“外显子”以找到第一个编码外显子和起始 ATG(图 3)。
注意:如果基因有替代转录本,请确保没有替代起始 ATG。如果有替代的起始密码子,目标是在所有转录保守生成强损失第一外显子-的-功能等位基因。


图 3. ensembl.org 基因组浏览器上斑马鱼hand2基因的外显子序列和下载页面

单击下载序列按钮(图 3,红色箭头)。的选择基因组,编码,非翻译区的列表,和内含子中示出的序列。选择 cDNA 和目标外显子并下载为单独的序列文件。
使用ApE < http://biologylabs.utah.edu/jorgensen/wayned/ape/ >或SnapGene ,用外显子注释编码序列。
设计引物以从鳍夹基因组 DNA 中扩增目标外显子并对扩增子进行测序。使用此序列手动识别 gRNA位点,通过搜索 PAM 序列,或使用CRISPRScan 。删除添加到 5' 端的额外“ Gs ” CRISPRScan 。选择一个š gRNA位置不具有在-帧ATG的下游š gRNA目标站点。在 cDNA 序列文件中注释选定的目标序列和 NGG PAM。
合成的gRNA可以从Synthego或 IDT订购。在Synthego订购页面上,选择“ 2'-O-Methyl at 3 first and last bases, 3' phosphorothioate bond between first 3 and last 2 bases ”“ Modifications”选项卡。
d ESIGN〜20个碱基的DNA引物为〜130的PCR扩增子bp的DNA的周围因组的靶位点。这些引物将用于在注射 CRISPR 试剂后从胚胎中扩增基因组 DNA,以测试目标位点的诱变。目标位点插入缺失的存在可以通过多种方式在 PCR 产物中检测到,包括凝胶电泳以可视化异源双链体的形成、在与sgRNA目标重叠的位点处对限制性内切酶消化的抵抗力以及直接测序,然后进行 ICE 分析。
用户小号可以用底漆3设计引物http://biotools.umassmed.edu/bioapps/primer3_www.cgi。
将目标站点周围的 DNA 序列粘贴到 Web 界面中。建议使用 160 - 300 bp的外显子序列,以切割位点为中心进行引物设计。可以使用内含子序列,但这通常包含可能导致扩增失败的多态性。
定位目标序列,包括 PAM 序列 NGG(在下面的示例中加下划线,粗体),并预测切割位点(此处由“x”表示的 PAM 上游3 bp )。纪念针对性的外显子序列约65 - 150个碱基对切削部位的两侧通过把[方括号,以黄色突出显示]周围。Primer3 将设计此序列之外的引物。这种设计允许引物在检查整合时用于检查诱变和连接片段分析。

例子:
CGGCCTCGGGATCCACCGGCC [ AGAATCGATATACTACGATGAACAGAGCAAATTTGTGTGTAATAC GGTCGCCACCATGGCCTxCCT CGG TTTGCTACGATGCATTTGCACCACTCTCTCATGTCCGGTTCTGGG ] AGGACGTCATCAAGGAGTTCATGCGCTTCAAGGTGCGCATGGAGGGCTCCGTGAAC

将“Primer Size”变量设置为 Min = 130、Opt = 170 和 Max = 300。其他一切都可以保留默认值。
单击“选择引物” 。”
从输出中选择引物。请注意预期的“产品大小”和每个引物/对的“tm”或熔解温度。中号utagenesis更容易显现以s小光伏产品尺寸。

SpCas9 mRNA的制备
用Xba1消化 ~5 - 10 μg pT3TS-nCas9n 质粒(质粒Addgene #46757 )(Jao 等人,2013 年)以线性化载体。
净化线性化DNA与一个Qiagen公司PCR净化试剂盒或Promega公司PureYield质粒Miniprep试剂系统。在洗脱RNA酶-f REE水。
在 1 × TAE 中的1.2% 琼脂糖凝胶上运行 100 - 500 ng,以确认质粒已线性化。
使用mMESSAGE mMACHINE T3 试剂盒 Life Technologies (AM1348)使用 100 ng 至 1 μg DNA 作为模板进行体外转录反应。按照套件随附的制造商说明进行操作。保存 1 ul等分的体外合成反应。
使用miRNeasy Qiagen试剂盒根据制造商的说明纯化nCas9n mRNA。
在miRNeasy Qiagen试剂盒清理之前和之后,通过在凝胶上运行体外合成的 mRNA样本来验证 mRNA 的完整性。混合 1 μ升Cas9的mRNA,4的μ升分子级RNA酶/ DNA酶的˚F稀土元素的水,和5 μ升乙二醛染料(NorthernMax -甘氨酸上样染料,热费舍尔,AM8551 )。
在 50 度加热混合物在水浴或热循环仪中保持°C 30 分钟,然后置于冰上。
清洁凝胶盒,梳子,并与托盘DECON ElIMINase和漂洗用DI水。
运行的所有10 μ升RNA混合物的上一个1.2%琼脂糖凝胶在1 ×如在100V TAE 1个小时描述的上方。图像凝胶我Ñ一个iBright FL1500成像系统或一个其他凝胶的文档成像系统。一个波段应该在 ~4.5 kb 处可见。
使用Nanodrop确定 RNA 样品的浓度。0.45和1之间的浓度微克/ μ升的预期。
在 -80°C 下分装并储存 RNA。

注射sgRNA和 spCas9 mRNA
在单细胞阶段向胚胎提供 2 n l体积的25 pg sgRNA和 300 pg Cas9 mRNA 。下面是斑马鱼胚胎注射的分步协议。
可以在 Rosen 等人中找到斑马鱼胚胎注射的详细视频。(2009 年)。用定制模具铸造斑马鱼胚胎注射托盘,创建 45 °槽,用于排列和保持胚胎(图 4A )。模具也有市售(见材料和试剂)。
熔融1.2%琼脂糖在1 × E2培养基倒入一个聚苯乙烯P ETRI菜。模具被设置在顶部(图4B ),并且,一旦所述板已经设置,轻轻取出模具(图4C )。进样盘可以多次使用,并在使用之间倒置在 4 °C下最多可存放三周。


图 4 .注塑托盘模具。注射盘模具( A )设置在熔化的 1.2% 琼脂糖 ( B ) 的顶部。带有槽的固化注射板以容纳胚胎 (C)。

P重新温暖注入托盘28.5 ℃,在注射前通过将它们放置在一个28.5 ℃的培养箱中20分钟。
在 Sutter Instrument Flaming/Brown Micropipette Puller 上使用 Kwik-Fill 硼硅玻璃毛细管拉出microcapillary玻璃针。
制备含有在分子级RNA酶/ DNA酶下文稀释注射样品˚F REE水:
12.5纳克/ μ升基因组的因组
150毫微克/ μ升用于Cas9 mRNA的
将注射液置于冰上
使用注射液后送针微加载枪头,并连接到一个显微。将持针器管连接到哈佛仪器 PLI-90 Pico 注射器。打开氮气或空气压缩机,以加压系统和一套喷射压力至40 PSI为100的喷射时间- 200毫秒。
通过首先用无菌镊子断开尖端的末端来校准注射针(图 5A、B)。使用踏板排出 10 个液滴并用 30 毫米长的毛细管捕获每个液滴,该毛细管的体积为 1 μ l (图 5C)。测量从毛细管的液体和转换的弯月面的端部的距离一体积的1毫米= 30 Ñ升; 因此,2/3 毫米 = 20 nl 。通过改变注射时间来调整每个液滴的体积,以达到 2 n l液滴s 。在设定压力下,体积和时间之间存在线性关系。避免注射时间降低超过100毫秒和高比400毫秒。
传送从收集一个细胞胚胎P ETRI菜槽上的预温热琼脂糖注入托盘(图5D)。每个胚胎都包裹在绒毛膜中。由于胚胎在受精后 45 分钟接近第一次细胞分裂,在蛋黄顶部清晰可见单个细胞(图 5D)。
使用显微操作器将针刺入绒毛膜并进入胚胎。在蛋黄界面/单细胞与蛋黄之间的边界处注入 2 n l样品(图 5E,白色箭头指向放置针尖的界面)。在第一次细胞分裂开始之前注射胚胎。
图5 。显微注射针校准和斑马鱼单-细胞胚胎显微注射。(甲)乙ackloaded注射针与封闭末端。(B)使用镊子去除针尖的一小部分以创建开口端。(C) 通过按下注射装置踏板排出一滴注射溶液。一个1的前端μ升曾用于捕获10滴德拉蒙德毛细管被示出。(d)ê mbryos在注射托盘槽与指示绒毛膜,蛋黄标签一字排开,和单-细胞胚胎。(E)ñ eedle通过绒毛膜和进入胚胎插入。注射针的尖端位于顶部的单细胞和下方的蛋黄之间的蛋黄界面(白色箭头)。第i (E)中法师发表在阿尔梅达等。(2021)。

注入后的离子,洗从注射托盘胚胎放入干净的P与胚胎媒体ETRI菜。
保持20 - 40个胚胎分离为使用未注射控制。
在3 - 5小时后注射,请从菜品的任何未受精或死胎。

试验因组诱变效率和插入缺失生产
双等位基因失活可导致可能致命的必需基因功能丧失。注射后,计数并从盘中取出死胚胎。如果所有胚胎都有缺陷且不太可能存活,则将注射的引导sgRNA量减少到 12.5 pg。如果胚胎仍然无法存活,则将 sgRNA的量进一步减少到 6.25 pg。正如我们前面,对于一个遍在表达,必需基因报道如肿瘤抑制RB1 ,注入的量因组需要被降低到6.25微克到恢复可行幼鱼存活至成年和发射系基因-编辑等位基因(索林等等,2015)。
胚胎消化以分离基因组 DNA 以进行诱变分析。
使用先前发布的以下协议(Wierson 等人,2020)从同一注射中的单个或五个胚胎池中提取基因组 DNA ,年龄在 1 到 5 dpf之间。
如果它们没有从绒毛膜中出现,则去除绒毛膜胚胎。
将胚胎放入 PCR 管中,并尽可能多地去除鱼水。收集至少三个注射胚胎和一个未注射的对照胚胎。
添加20 μ升50 mM的氢氧化钠每个胚胎。  
在热循环仪中将胚胎在 95 °C 下加热30 分钟。
涡旋样品并向下旋转管子。胚胎应完全溶解。 
通过加入1中和样品μ升的1M的的Tris pH 8.0中每10 μ升的NaOH 。通过涡旋混合,然后向下旋转。
基因组 DNA 储存在 -20 °C 。
分析目标基因位点的 CRISPR/Cas9 诱变效率。
从-20 °C 中取出基因组 DNA 样本并放在冰上解冻。始终将解冻的基因组 DNA 放在冰上。
设置以下PCR反应用于胚胎的每个管使用消化的基因组DNA的基因-特异性正向和反向引物的是被设计成创建CRISPR / Cas9目标部位周围的扩增子。
12.5 μ l 2 × GoTaq Mastermix
1 μ l正向引物 (10 μM )
1 μ升反向引物(10 μM )
1 μ升的的gDNA模板(消化胚胎)
9.5 μ升的无核酸酶水
对于一个25 μ升总
轻弹管子以混合并短暂地降低 PCR 反应速度。
运行以下 PCR 程序以放大目标基因座。
95 °C 2 分钟
95 °C 30 秒           
55 °C 30 秒35 个循环               
72 °C 30 秒           
72 °C 5 分钟                                       
4 °C保持             
运行多达7 μ升PCR产物上的2.5〜3%的琼脂糖凝胶在1 × TAE,在80-100V 1个小时。图像凝胶在iBright FL1500成像系统或一个其他凝胶的文档成像系统。 
的一例因组注射和验证靶向的外显子1 HAND2基因被示出在图6A中。对照未注射胚胎 PCR 扩增子在凝胶上作为单个紧密带运行(图 6B、U)。从扩增子8周注射的胚胎显示多个频带或在外观上漫相对于对照(图6B中,1 - 8)。这表明在感兴趣的基因的 CRISPR 靶位点存在indel突变导致 PCR 产物中形成异源双链体。


图 6. hand2外显子 1 sgRNA验证。一个。小号的层序的HAND2反向链因组位于外显子1 B位。PCR 扩增子与sgRNA目标位点侧翼的引物。在注射的胚胎漫射条带代表异源双链体引起的DNA插入缺失在靶位点处的突变。

对于诱变效率的定量分析,Sanger 对 PCR 产物进行测序以验证插入缺失的存在。的比例插入缺失的形成可以使用分析Synthego的ICE分析。或者,Illumina的MiSeq复下-代测序可用于测试并联的多个gRNAs的效率。
图 6 中hand2外显子 1 靶向胚胎 #3 和 #6的扩增子的 ICE 分析示例如下图 7 所示。胚胎 #3 和 #6在靶向后分别显示 84% 和 80% 的indel等位基因,表明高sgRNA 的诱变效率。


图 7.通过 ICE 分析验证sgRNA诱变效率。对来自hand2外显子 1 靶向胚胎 #3 (A) 和 #6 (B) 的PCR 扩增子进行 Sanger 测序,并使用Synthego ICE 分析对结果进行分析,显示 84% 和 80% 的序列包含indel突变。右侧的图显示了恢复的indel突变的范围。

设计用于pGTag靶向载体组装的短同源臂
同源性-定向基因靶向允许将外源 DNA 无缝整合到基因组中,并在目标位点进行精确的修复事件。然而,为每个感兴趣的基因设计和克隆单独的靶向载体和同源臂可能非常耗时。该pGTag载体系列和网页设计工具提供了多功能性和易于产生敲-出的等位基因(图8 )。载体包含BfuAI和BspQI II 型限制性内切酶,用于使用 Golden Gate 克隆来克隆短同源臂(24 或 48 bp )。该pGTag载体需要架集成适当报告基因的功能。报告基因由几个部分组成。2A 肽序列导致翻译跳跃,允许以下蛋白质从基因座肽解离。所述的eGFP ,TagRFP ,或Gal4VP16记者编码序列具有几个用于定位信号,包括胞质(无信号)的选择,核定位信号(NLS),或膜定位CAAX序列。最后,翻译是由两个不同的转录终止多聚腺苷酸化(之一终止pA的)序列,斑马鱼的3'UTR区域肌动蛋白基因或SV40病毒转录终止序列。
对于许多基因,内源基因表达水平不足以从整合的报告基因产生可检测的荧光信号。Gal4VP16 pGTag载体与转基因 Tol2<14XUAS/RFP> 报告系(Balciuniene 等人,2013 年)相结合,可以放大信号。Tol2<14XUAS/RFP> 报告线可应天普大学Darius Balciunas博士实验室的要求提供。
质粒序列图可从www.genesculpt.org/gtaghd/下载。


图8 。所述pGTag载体允许同源臂中的一个步骤的克隆。

所有载体(图8 )均可通过Addgene (www.addgene.org)获得。由于pGTag质粒包含重复序列,因此细菌中可能会发生载体重组。我们推荐使用NEB Stable Competent E.coli (New England Biolabs , C3040I)。细菌应在 30 °C下生长,以进一步减少载体重组。           

GTagHD 的同源臂设计
网络工具GTagHD允许快速设计寡核苷酸以生成与感兴趣基因中的目标位点互补的短 24 或 48 bp同源臂。
两个具有突出端的互补寡核苷酸退火生成双链同源臂,用于克隆到pGTag载体中。
要使用该工具,请选择“提交单个作业”选项卡。按照选项卡中的说明进行操作。
将返回两对互补寡核苷酸的序列,一对用于 5' 同源臂,另一对用于 3' 同源臂。如果输入的序列和值有问题,网页将显示错误和如何修复它们的建议。双重-检查你的输出如下。
手动同源臂设计
以下协议描述了如何手动设计同源臂寡核苷酸:
注意:在下面的部分中,目标位点和同源性的方向是在感兴趣的基因座的阅读框架的背景下。示例:正向链 CRISPR gRNA意味着gRNA 和 PAM在基因的有义链上编码。上游同源性结构域的5'到所述CRISPR / Cas9切割位点,和下游同源结构域3'到切割位点相对于所述的的读码框的基因作为目标。
还要注意:大写和小写的基数没有特别修改;这只是同源臂不同部分的视觉标记。

上游同源臂设计
打开感兴趣的基因序列文件,并确定CRISPR网站(我此实例,它是黄河反向CRISPR目标时,PAM为橙色,而编码序列是紫色)。将CRISPR 切割的 48 bp 5'(下面突出显示的部分)复制到一个新的序列文件中;这是上游同源性(图 9)。
图9 。ApE中显示的目标基因的屏幕截图,突出显示目标序列(黄色)、PAM(橙色)、编码序列(紫色)和上游同源臂的基因序列(突出显示白色)。

观察紧邻 48 bp同源序列上游的三个碱基,并选择一个不存在的碱基作为同源序列和载体中通用 PAM 之间的3 bp间隔区。在这里,三个基地是“GGA ,”所以选择“CCC”为间隔。将间隔添加到同源的新文件 5'(前面)(见下文)。间隔区充当同源性和来自通用引导序列的最终 6 bp皮瓣之间的非同源缓冲液,当盒带被释放时,该序列将发生,并且可能会提高 MMEJ 事件的预期整合率(图 10)。
图10 。上游同源臂(紫色)、PAM(橙色)和切割位点的剩余目标序列(黄色)的基因序列的屏幕截图。CCC加入作为与间隔一非同源序列。

确定最后一个密码子在同源性中的位置。此处,同源域中的 3' G 是此 CRISPR 靶标切割的密码子中的第一个碱基。通过添加序列中该密码子的剩余碱基(在GTagHD上称为填充)来完成密码子,以确保您的整合事件在框架内(图 11)。
图11 。上游同源臂的基因序列(紫色)、PAM(橙色)和带有填充核苷酸 ( tg )的切割位点的剩余目标序列的屏幕截图,以保持框架内的整合(黄色)。

将用于克隆的BfuAI酶悬垂序列添加到同源域的末端。这里,这两个突出端,5'- GCGG和3'-GGAT ,被添加以防止在用于复制的序列错误的寡核苷酸在接下来的两个步骤(图12)。
图12 。上游同源臂(紫色)、PAM(橙色)的基因序列的屏幕截图,以及带有填充核苷酸 ( tg )的切割位点的剩余目标序列,以保持框架内的整合(黄色)并添加了BfuAI位点到每一端。

上游同源寡核苷酸 A 将是从最后一个密码子的开头到结尾的序列(参见下面突出显示的部分)。将此序列复制并粘贴到新文件中并保存。在这个例子中,这个寡核苷酸序列是 5'-GCGGcccGTTTTCTTACGCGGTTGTTGGATGAAATCTCCAACCACTCCACCTTCGtg-3' (图 13)。
图13 。上游同源臂(紫色)、PAM(橙色)的基因序列的屏幕截图,以及带有填充核苷酸 ( tg )的切割位点的剩余目标序列,以保持框架内的整合(黄色)并添加了BfuAI位点到每一端。Oligo A 的序列为白色。

上游同源寡聚乙将从该序列的反向互补的间隔到该序列的结束的开始(见图强调下面14 )。复制反向补码,将其粘贴到新文件中,然后保存。在这个例子中,这个寡核苷酸序列是 5'-ATCCcaCGAAGGTGGAGTGGTTGGAGATTTCATCCAACAACCGCGTAAGAAAACggg-3'。
图14 。上游同源臂(紫色)、PAM(橙色)的基因序列的屏幕截图,以及带有填充核苷酸 ( tg )的切割位点的剩余目标序列,以保持框架内的整合(黄色)并添加了BfuAI位点到每一端。Oligo B 的序列被突出显示。使用突出显示序列的反向补码。

下游同源臂设计
打开感兴趣基因的序列文件并确定 CRISPR 位点。将R EVERSE CRISPR目标是在黄海,在奥兰治PAM,和紫色的编码序列。将CRISPR 剪切的 48 bp 3'复制到新的序列文件中;这是下游同源性(图 15)。
图15 。靶基因的屏幕截图,突出的靶序列(黄色),PAM(橙色),编码序列(紫色)和下游同源臂的基因序列(在白色)。

观察 48 bp同源性下游的下三个碱基,并选择一个不存在的碱基作为同源性和载体中通用 PAM 之间的3 bp间隔区。这里,基数是“CTG ; ”因此,选择“ aaa ”作为间隔物。将垫片添加到同源(之后)的新文件 3' (图 16)。
图16 。在该基因序列的屏幕截图的来自目标基因下游同源臂。这包括部分目标序列(黄色)和额外的 3' 编码序列(紫色)。添加aaa作为填充核苷酸。

将用于克隆的BspQI酶悬垂序列添加到同源域的末端。这里,这两个突出端,5'-AAG和3'-CCG ,被添加以防止复制的序列错误的寡核苷酸在接下来的两个步骤(图17)。
图17 。来自目标基因的下游同源臂中基因序列的屏幕截图,其中包含部分目标序列(黄色)和额外的 3' 编码序列(紫色)。BspQI酶突出端序列添加到每个末端。

下游同源寡核苷酸 A 将是这个序列,从序列的开头到间隔的结尾(参见下面突出显示的部分)。在这个例子中,这个寡核苷酸序列是5'-AAGTGGCAAGATATGGCTCACGTTATTCATCATCTTCCGCATTGTTTTGAaaa-3' (图 18)。
              图18 。来自目标基因的下游同源臂中基因序列的屏幕截图,其中包含部分目标序列(黄色)和额外的 3' 编码序列(紫色)。Ť他小号的寡一个层序是白色的。

下游同源寡核苷酸 B(将是该序列从同源性开始到序列结束的反向互补(见下文突出显示)。在本例中,该寡核苷酸序列是5'- CGGtttTCAAAACAATGCGGAAGATGATGAATAACGTGAGCCATATCTTGCCCA-3' (图 19) .
图19 。来自目标基因的下游同源臂中基因序列的屏幕截图,其中包含部分目标序列(黄色)和额外的 3' 编码序列(紫色)。在小号的寡乙层序以白色高亮显示。应订购反向补充。

正确同源臂设计的一个示例,显示了用于克隆到pGTag和pPRISM BfuAI和 BspQ1 位点以用于hand2 gRNA 位点的互补突出端(图 20)。
图 20. pGTag和pPRISM载体同源臂设计示例,显示了用于克隆到BfuAI和BspQI II 型限制酶位点的互补 5' 突出端。hand2基因中的 CRISPR/Cas9 靶位点图。gRNA SEQ UE NCE红色和PAM序列下划线和粗体。退火的同源臂寡核苷酸A 和 B 显示有与酶消化后载体突出端互补的突出端(绿色)。n ,间隔核苷酸;n. ,包括核苷酸以维持pGTag整合等位基因的阅读框。


将同源臂一锅克隆到pGTag载体中
ñ OTE S:
如果同源臂寡核苷酸包含 5'-ACCTGC-3' 或 5'-GAAGAGC-3'序列(或它们的互补序列),克隆反应的效率将降低。
如果一个图形克隆不成功,5'和3'同源臂可顺序地使用凝胶克隆到载体-线性用适当的内切酶消化的质粒进行纯化。
同源臂退火
分别退火上下游同源寡核苷酸对:
              4.5 μ l oligo A,浓度为 10 μM
              4.5 μ l olio B,浓度为 10 μM
              4 μ l 10 ×缓冲液 3.1,来自 NEB
              27 μ升卫生署2 ö
对于一个40 μ升总           
以退火的寡核苷酸,运行在热循环仪下面的程序:第1步,孵化,在98 ℃下为5英里nutes ; 第二步,97 ℃孵育45秒;重复步骤 2 90 个循环,期间温度降低 1 °C/循环;最后一步保持在4 °C 。或者,将玻璃烧杯中的水在加热板上煮沸,然后将试管在沸水中孵育5分钟。ř EMOVE从热源烧杯并让其冷却至室温erature。将退火的同源臂储存在冰上或 -20 °C 的冰箱中。
1-Pot 摘要
混合以下解决方案:
              4.0 μ升卫生署2 ö
              2 μ l质粒,浓度为 50 ng/ μ l
              1 μ l 10 ×缓冲液 3.1,来自 NEB
              1 μ l 5' 退火同源臂
              1 μ l 3' 退火同源臂
              0.5 μ升BfuAI自NEB酶
              0.5 μ升BspQI自NEB酶
              对于一个10 μ升总
50 °C孵育1 小时,置于冰上。
结扎
添加以下内容:
                                                        3 μ l 5 × T4 快速连接酶缓冲液
              1.5 μ升卫生署2 ö
              0.5 μ升T4连接酶快
              对于一个15 μ升总
在室温下孵育 8-10 分钟(至过夜)。储存在-20 °C 。
转化:为了防止重组在质粒重复元件,在30生长转化和过夜培养℃下。我们的标准协议使用 NEB 稳定感受态大肠杆菌(C3040H) 细胞来克隆和繁殖GeneWeld pGTag和pPRISM质粒系列以限制重组。
在冰上,解冻1(一)的小瓶感受态细胞(50 μ升)每2个连接反应(约5分钟)。
当细胞解冻时,为每个结扎标记微量离心管并放在冰上。
一旦细胞解冻,用移液管向25转移μ升的感受态细胞到每个标记的管中。 
添加1.5 μ升连接反应成感受态细胞转化的。
的一个安装件添加数量应小于5%的连接反应的的感受态细胞的体积。
通过轻敲管子数次或用移液器吸头轻轻混合来混合。
注意:不要通过移液混合;这将溶解细胞。
在冰上孵育 5 到 20 分钟。
热-通过浸入含有在42细胞中的管的该部分震荡细胞℃的水浴中40 - 50秒。
在冰上孵育细胞 2 分钟。
添加125 μ升室温LB或SOC的每个变换。
在 30 °C下在摇动孵化器中孵育细胞1-1.5 小时。
而转化的细胞回收,扩展40 μ升的X-Gal的溶液和40 μ升IPTG 0.8 M于LB卡那霉素选择平板。 
X-Gal 在潮湿时对细胞是致命的;建议先给板贴上标签,然后将它们放入 30 °C 的培养箱中晾干。
后的恢复和X-Gal的是干的,p晚150 μ升上相应的正确标记的板中的每个变换。
在 30 °C 下过夜孵育板。
不断增长的殖民地
挑3个从每个板的白色菌落,并在单独的玻璃培养管生长带3米升在30 LB /卡那霉素,过夜℃下,邻- [R通过菌落PCR进行预先筛选的菌落:
拾取到8个菌落用移液管尖端和重悬在它们的5等份分开μ升卫生署2 O.将在3ml LB /尖端阚在4,标签,和存储℃下。
为您的 PCR 反应制作一个预混液,其中包含以下数量乘以您挑选的菌落数。
7.5 μ l 2 × GoTaq mastermix
5.5 μ升卫生署2 ö
0.5 μ升引物在10 μ中号“ F3'检查” 5'-GGCGTTGTCTAGCAAGGAAG-3'
0.5 μ升引物在10 μ中号“3'_pgtag_seq” 5'-ATGGCTCATAACACCCCTTG-3'
对于一个14 μ升总
等分试样14 μ升混合主混合物的成单独的标记的PCR管中。
加入1 μ升菌落的各反应作为模板,或20纳克纯化的质粒作为对照。
在热循环仪中循环
95 °C 2 分钟           
95 °C 30 秒             
57 °C 30 秒35 个循环               
72 °C 30 秒           
72 °C 5 分钟             
4 °C保持             
运行5 μ升在1%琼脂糖凝胶PCR产物。使图像凝胶iBright FL1500成像系统或一个其他凝胶的文档成像系统。应该有与对照不同大小的条带。
迷你准备文化
              遵循Qiagen协议
质粒测序
5' 同源臂可以通过 5'_pgtag_seq 引物进行测序:
5'-GCATGGATGTTTTCCCAGTC-3'。
3'同源臂可以用“3'_pgtag_seq”引物测序:
5'-ATGGCTCATAACACCCCTTG-3'。

的注射GeneWeld试剂(spCas9的mRNA,环球因组(UgRNA ),基因组因组,并pGTag同源性载体中)进入1细胞斑马鱼胚胎
准备和收集以下注射试剂:
从 pT3TS-nCas9n ( Addgene #46757 ) (Jao et al., 2013) (见B 部分 5. SpCas9 mRNA 的制备)制备 nCas9n mRNA 。
的UgRNA和基因组因组可直接序F - [R OM IDT或Synthego和再悬浮在分子级RNA酶/ DNA酶˚F稀土元素的水。
所述pGTag同源性向量应该之前显微注射用无RNA酶的条件下进行纯化的第二时间Promega公司PureYield质粒Miniprep试剂系统在去除内毒素洗涤开始。质粒 DNA 在分子级 RNase/DNase-Free 水中洗脱。
用于整合pGTag载体的胚胎注射。
以每个胚胎2 nl的体积注射单细胞胚胎,其中含有以下浓度的 RNA 和载体:
在注射液中在胚胎中                         
75 pg / nl nCas9n mRNA 150 pg nCas9n mRNA                                                                                               
12.5 pg / nl基因组sgRNA 25 pg基因组sgRNA                                                                     
12.5皮克/ NL的UgRNA 25微克的UgRNA                                                                                                 
5微克/ NL的pGTag DNA  10微克的pGTag DNA                                                                                 

测试注射的胚胎以证明精确的目标整合
在蔡司 Discovery 解剖显微镜下检查注射胚胎的荧光,放大率为1 ×物镜,放大率为 70-100 × 。如果观察到微弱的信号,手动dechorionate胚胎,并在带有落射照明的标准立式复合显微镜上的玻璃凹陷井幻灯片上查看。也可以进行高分辨率共焦实时成像,如Wierson等人所示。(2020 年)图 3 (https://elifesciences.org/articles/53968/figures#fig3)。
用于荧光蛋白激活的光源类型会显着影响荧光信号的可视化能力。带有光纤电缆的 X-Cite 120W 金属卤化物光源 ( Excilitas Technologies) 可以很好地显示体细胞靶向后的荧光。所述TagRFP蛋白也被移位在其激发,并且该蛋白优化过滤器推荐。还建议使用针对 GFP 和 BFP 优化的过滤器。如果没有观察到信号或观察到弱信号,pGTag-Gal4VP16 的整合可用于放大 14XUAS-RFP 转基因系中的报告基因表达(Balciuniene等,2013 )。
对在与目标基因一致的表达域中显示广泛荧光的阳性胚胎进行连接片段 PCR 分析。从单个胚胎和对照胚胎中分离基因组 DNA (参见D 2. 胚胎消化以分离基因组 DNA 用于诱变分析)。PCR扩增基因组 DNA 整合盒 5' 和 3'连接片段。F0胚胎结分析的一个例子示于图3S1在Wierson等。, 2020 ( https://elifesciences.org/articles/53968/figures#fig3s1 )。
以下引物用于连接片段分析,必须与基因配对-特异性引物(5'至3' ):
5' pGTag连接点:
R-Gal4-5'juncM GCCTTGATTCCACTTCTGTCA和基因-特异性正向引物
R-RFP-5'jun c CCTTAATCAGTTCCTCGCCCTTAGA
R-eGFP-5'-jun c GCTGAACTTGTGGCCGTTT
3' pGTag连接点:
F-Gal4-3'juncM GCAAACGGCCTTAACTTTCC 和基因特异性反向引物
F-Gal4-3'junc CTACGGCGCTCTGGATATGT
F-RFP-3'junc CGACCTCCCTAGCAAACTGGGG
F-eGFP-3'jun c ACATGGTCC TGCTGGAGTTC

为了控制(Won 和 Dawid,2017 年)中描述的 PCR 扩增伪影,对注射了所有靶向试剂减去基因组sgRNA 的胚胎进行 PCR 连接分析。
替代引物 F-Gal4-3'juncM 和 F-Gal4-3'juncJ 可能会增加引物特异性,具体取决于目标基因。
7.5 μ l 2 × GoTaq mastermix
5.5 μ升卫生署2 ö
0.5 μ升引物在10 μM的基因组的引物
0.5 μ升在10底漆μM pGTag底漆
对于一个14 μ升总
等分试样14 μ升混合主混合物的成单独的标记的PCR管中。
加入1 μ升的基因组DNA的各反应作为模板。
通过以下步骤在热循环仪中循环:
              95 °C 2 分钟             
              95 °C 30 秒           
              55 °C 30 秒35 个循环               
              72 °C 30 秒                           
              72 °C 5 分钟           
              4 °C保持             
运行5 μ升的在1在1.2%琼脂糖凝胶的PCR产物× TAE。使图像凝胶的iBright FL1500成像系统或一个其他凝胶的文档成像系统。假定的连接片段应提供具有预测大小的条带。

建立新的精准靶向整合等位基因转基因品系
提高到成年荧光记者-表达注射胚胎的 F0 兄弟姐妹,在5' 和 3' 交界处分析显示阳性带,表明精确靶向整合。Outcross F0 成虫与野生型并检查报告基因荧光的后代,如上文所述,以确定继承了稳定种系整合等位基因的 F1 胚胎。对于 Gal4Vp16 整合等位基因,将 F0 成虫与 14XUAS :RFP记者线交叉。14XUAS 的沉默:RFP报告基因可能导致 Gal4Vp16 靶向 F1 胚胎中的镶嵌表达模式。
如上所述,通过连接片段 PCR 分析测试 F1 荧光阳性胚胎以进行精确的转基因整合。加注F1兄弟姐妹到成年和鳍形夹为示出了与精确靶向的转基因整合识别个体图4S 2 - 4的Wierson等。(2020 年)(https://elifesciences.org/articles/53968/figures#fig4s2)。
与单个阳性 F1 成人杂交以建立 F2 家庭。F1s的也可以牺牲后3周-施肥确认位置和有针对性的整合的精度通过基因组Southern印迹RFLP分析如在图4S1的Wierson等。(2020 年)(https://elifesciences.org/articles/53968/figures#fig4s1)。在后代中通过与野生型异交继续保持品系。表1和2的Wierson等。( 2020 ) ( https://elifesciences.org/articles/53968/figures )显示了精确靶向整合等位基因在八个斑马鱼位点的种系传输频率范围。
要进行有针对性的整合等位基因是否在功能表型的损失进行初步评估,F0和F1鉴定鱼类可以incrossed或交叉到已知的插入缺失靶向基因的等位基因。

数据分析

原始文章( Wierson et al. , 2020 )中数值数据的链接包含在协议中。

食谱

斑马鱼胚胎 E2 培养基(Westerfield, 1995) ( https://wahoo.cns.umass.edu/book/export/html/867 )

致谢

这项工作是由支持的美国国立卫生研究院资助R24OD020166(JJE,MM,DD,司法委员会,以及SCE) 。本出版物中报告的研究部分得益于堪萨斯大学基因组测序核心实验室的服务,该实验室由 NIH 国家普通医学科学研究所 (NIGMS) 提供支持,奖项编号为 P20GM103638。所述GeneWeld协议与相关联Wierson等。, 2020 年。

利益争夺

JJE、MM 和 KJC 与Recombinetics , Inc.存在财务利益冲突;JJE 和 SCE 与Immusoft , Inc.;JJE,MM,WAW,司法委员会,并与SCE LifEngine和LifEngine动物技术。

伦理

本协议中描述的所有斑马鱼实验均根据爱荷华州立大学动物护理和使用委员会日志 #11-06-6252 的批准协议进行,符合美国兽医协会和 NIH 研究中人道使用动物的指南。

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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Welker, J. M., Wierson, W. A., Almeida, M. P., Mann, C. M., Torrie, M. E., Ming, Z., Ekker, S. C., Clark, K. J., Dobbs, D. L., Essner, J. J. and McGrail, M. (2021). GeneWeld: Efficient Targeted Integration Directed by Short Homology in Zebrafish. Bio-protocol 11(14): e4100. DOI: 10.21769/BioProtoc.4100.
  2. Wierson, W. A., Welker, J. M., Almeida, M. P., Mann, C. M., Webster, D. A., Torrie, M. E., Weiss, T. J., Kambakam, S., Vollbrecht, M. K., Lan, M., McKeighan, K. C., Levey, J., Ming, Z., Wehmeier, A., Mikelson, C. S., Haltom, J. A., Kwan, K. M., Chien, C. B., Balciunas, D., Ekker, S. C., Clark, K. J., Webber, B. R., Moriarity, B. S., Solin, S. L., Carlson, D. F., Dobbs, D. L., McGrail, M. and Essner, J. (2020). Efficient targeted integration directed by short homology in zebrafish and mammalian cells. Elife 9: e53968.
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