基因君官网
2015年6月9日讯 /生物谷BIOON/ –近日，来自美国NIH的研究人员进行了一项研究，他们利用CRISPR-CAS9技术靶向斑马鱼特定DNA序列进行基因功能探索和人类治病基因的发现研究，相关研究成果在线发表在国际学术期刊genome research。
在这项研究中，研究人员发现利用基因编辑技术CRISPR-CAS9进行斑马鱼基因靶向识别以及插入删除特定基因的效率可以达到其他技术的6倍。同时，利用CRISPR-CAS9技术还能够在同一时间对多个基因进行靶向突变，为研究基因功能提供了极大便利。
研究人员指出，斑马鱼是一种常用的实验动物模型，并且人类已经完成对斑马鱼的基因测序，由于斑马鱼有70%的基因与人类同源且斑马鱼的繁殖快，产量大，并且饲养更加经济，因此相比于其他模式动物，斑马鱼更适合进行高通量筛选。
在该项研究中，研究人员为探究利用斑马鱼进行高通量基因编辑的可行性，他们利用CRISPR－CAS9技术对83个斑马鱼基因的162个位点进行了基因编辑，其中有大约50%的基因与人类耳聋相关基因类似。最终通过这种方法，共获得82个基因的突变。随后研究人员又利用荧光PCR和高通量DNA测序的方法对斑马鱼的胚胎进行了筛选，他们发现有大约28%的突变能够遗传给下一代。
这项研究表明，利用CRISPR－CAS9进行斑马鱼基因的编辑有可能实现对斑马鱼基因组所有基因进行突变，随后进行大规模表型筛选，这对于基因功能的探索以及人类致病基因的发现都具有重要意义。（基因宝jiyinbao.com）
High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9
Gaurav K. Varshney1, Wuhong Pei1, Matthew C. LaFave1, Jennifer Idol1,5, Lisha Xu1, Viviana Gallardo1, Blake Carrington1, Kevin Bishop1, MaryPat Jones2, Mingyu Li3, Ursula Harper2, Sunny C. Huang1,6, Anupam Prakash1, Wenbiao Chen3, Raman Sood1, Johan Ledin4 and Shawn M. Burgess
The use of CRISPR/Cas9 as a genome-editing tool in various model organisms has radically changed targeted mutagenesis. Here, we present a high-throughput targeted mutagenesis pipeline using CRISPR/Cas9 technology in zebrafish that will make possible both saturation mutagenesis of the genome and large-scale phenotyping efforts. We describe a cloning-free single-guide RNA (sgRNA) synthesis, coupled with streamlined mutant identification methods utilizing fluorescent PCR and multiplexed, high-throughput sequencing. We report germline transmission data from 162 loci targeting 83 genes in the zebrafish genome, in which we obtained a 99% success rate for generating mutations and an average germline transmission rate of 28%. We verified 678 unique alleles from 58 genes by high-throughput sequencing. We demonstrate that our method can be used for efficient multiplexed gene targeting. We also demonstrate that phenotyping can be done in the F1 generation by inbreeding two injected founder fish, significantly reducing animal husbandry and time. This study compares germline transmission data from CRISPR/Cas9 with those of TALENs and ZFNs and shows that efficiency of CRISPR/Cas9 is sixfold more efficient than other techniques. We show that the majority of published “rules” for efficient sgRNA design do not effectively predict germline transmission rates in zebrafish, with the exception of a GG or GA dinucleotide genomic match at the 5′ end of the sgRNA. Finally, we show that predicted off-target mutagenesis is of low concern for in vivo genetic studies.