Transient CRISPR-Cas9 targeting of the alcama and pdgfra genes was performed by using Alt-R CRISPR-Cas9 system (Integrated DNA Technologies / IDT). The gRNA target sites (shown in Supplementary Table 10 ) were evaluated for the off-target sites by using the sgRNA design tool (IDT). Alt-R S.p. Cas9 V3 enzyme (IDT) and equimolar amount of crRNA:tracrRNA duplexes (IDT) were mixed and incubated to form RNP complexes before injection. The mixed solution was diluted with Cas9 buffer (NEB) to adjust the concentration of Cas9 and sgRNA working solution to 12 and 18 µM, and 1 nl was injected into the cytoplasm of one-cell stage Tg(pax3a:eGFP; kdrl:ras-mCherry) embryos. Injected F0 crispant embryos were individually screened by genomic PCR using primers that recognize outer sites of the region sandwiched by a pair of gRNA (for alcama) or primers designed at intron2/exon3 of the pdgfra gene. For the cDNA analysis of pdgfra-crispants, total RNA was extracted from 100 to 120 pooled embryos and RT-PCR was performed using primers designed at exon2/exon4 to check the resulting splicing patterns. The primers used are shown in Supplementary Table 10 .
Cas9 buffer
Manufactured by New England Biolabs
Sourced in United States
Cas9 buffer is a solution designed to provide an optimal environment for the activity of the Cas9 enzyme, a key component of the CRISPR-Cas9 genome editing system. The buffer's core function is to maintain the appropriate pH, ionic conditions, and cofactors necessary for Cas9 to effectively bind and cleave target DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Alt r crispr cas9 system, by Integrated DNA Technologies (1 mentions)
Cas9 protein, by New England Biolabs (1 mentions)
Phosphor imaging cassette, by Bio-Rad (1 mentions)
Phosphor imaging cassette, by GE Healthcare (1 mentions)
Personal molecular imager, by Bio-Rad (1 mentions)
Mmessage mmachine sp6 transcription kit, by Thermo Fisher Scientific (1 mentions)
4 protocols using cas9 buffer
1
CRISPR-Cas9 Targeting of alcama and pdgfra
2
CRISPR Targeting of abl1 and abl2 in Zebrafish
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gRNAs targeting the exons of both abl1 and abl2 genes were designed using CHOPCHOP website [52 (link)] (https://chopchop.cbu.uib.no/ ) and were synthesized from Synthego Inc, using Synthego modified EZ Scaffold option. The sequence of gRNAs is shown in S1 Table . All six gRNAs were added in equal amounts to the final concentration of 6 μM of total gRNA and 4 μM Cas9 protein and 1x Cas9 buffer from New England Biolabs (Cat No. M0646T). The reaction mixture was incubated at 37°C for 5 min and allowed to cool down to room temperature before performing microinjections. Approximately 2 nl of Cas9/gRNA mixture was injected into a blastomere of each kdrl:GFP embryo.
3
Quantitative Cas9 Cleavage Assay
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For Cas9 Cleavage reactions 33 nM Cas9 (NEB), 120 nM sgRNA and 1× Cas9 buffer (NEB) were pre-incubated for 20 min at 37°C. Subsequently 5′ 32P-radiolabeled target DNA was added to a final concentration of 3 nM and the reaction was incubated for 2 h at 37°C. 30 μl 2× formamide loading dye (95% formamide, 0.125% bromophenol blue) was added and the complete samples were heated to 95°C for 5 min and loaded on an 8% acrylamide gel (with 7 M urea and 1× TBE). The gel was run in 1× TBE for ∼4 h at 15 mA and subsequently exposed for 16–48 h in a phosphor imaging cassette (Molecular Dynamics) at –20°C. The phosphor imaging cassette was scanned using a Personal Molecular Imager (Bio-Rad).
4
CRISPR Targeting of Zebrafish rp1l1
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CRISPR sites were identified in the first coding exon of rp1l1 (Gene ID: 101882236) using Geneious R9 (Biomatters Inc., San Diego, CA, USA). Previously, there were two rp1l1 homologues annotated in zebrafish: rp1l1a and rp1l1b. However, rp1l1b did not resemble rp1l1 genes in other organisms (including humans, mice, cows, pigs, dogs, chickens, and Xenopus), as it was missing defining features that identify rp1l1 and was almost twice the size. In addition, in the current zebrafish genome (z10), rp1l1b is no longer annotated. Therefore, it appears that zebrafish have one rp1l1 homolog, formerly annotated as rp1l1a, which we targeted for our experiments. The guide RNA (gRNA; sequence: 5′-CATCTTGACGCCTTTGAACT-3′) was synthesized as previously described [18 (link)], using the mMessage mMachine SP6 Transcription Kit (Invitrogen, Waltham, MA, USA). Embryos were injected at the single-cell stage using a glass needle mounted on a micromanipulator. The injection mix consisted of 1 μL (>1500 ng/μL) of gRNA, 2 μL of Cas9 nuclease, S. pyogenes (New England Biolabs, Ipswich, MA, USA), 0.5 μL of Cas9 buffer (New England Biolabs, Ipswich, MA, USA), and 1.5 μL of 1.5 M KCl.
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