Inconsistent activity limits the use of CRISPR-Cas9 in zebrafish. We show supernumerary guanine nucleotides at the 5' ends of single guide RNAs (sgRNAs) account for diminished CRISPR-Cas9 activity in zebrafish embryos. Genomic sequences can be targeted consistently with extremely high efficiency using Cas9 ribonucleoproteins (RNPs) containing either a sgRNA molecule or a synthetic crRNA:tracrRNA duplex that perfectly matches the protospacer target site. Following injection of zebrafish eggs with such RNPs, virtually every copy of a targeted locus harbors an induced indel mutation. Loss of gene function is often complete, as F0 embryos closely resemble true null mutants without detectable non-specific effects. Mosaicism is sufficiently low in F0 embryos that cell non-autonomous gene functions can be probed effectively and redundant activities of genes can be uncovered when two genes are targeted simultaneously. Finally, heritable deletion mutations of at least 50 kbp can be readily induced using pairs of duplex guide RNPs targeted to a single chromosome.

中文翻译：

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高效的基于CRISPR-Cas9的方法来产生缺失突变和缺乏斑马鱼基因功能的F0胚胎。

不一致的活性限制了CRISPR-Cas9在斑马鱼中的使用。我们显示在单个向导RNA（sgRNAs）的5'端多余的鸟嘌呤核苷酸说明斑马鱼胚胎中的CRISPR-Cas9活性降低。使用含有sgRNA分子或完全匹配原型间隔位点的合成crRNA：tracrRNA双链体的Cas9核糖核蛋白（RNP），可以始终如一地高效靶向基因组序列。用这种RNP注射斑马鱼卵后，几乎每个目标基因座拷贝都具有诱导的indel突变。基因功能的丧失通常是完全的，因为F0胚胎非常类似于真正的无效突变体，而没有可检测到的非特异性作用。在F0胚胎中，镶嵌作用足够低，因此可以有效地探测细胞非自主基因的功能，并且当同时靶向两个基因时，可以发现基因的多余活性。最后，使用成对的靶向单个染色体的双链指导RNPs对，可以容易地诱导出至少50kbp的可遗传的缺失突变。