利用CRISPR/Cas9和piggyBac实现果蝇基因组无缝编辑

Seamless genome editing in Drosophila by combining CRISPR/Cas9 and piggyB ac technologies

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein9)是第三代基因组编辑技术。在sgRNA引导下,Cas9核酸内切酶作用于特定基因组序列,产生DNA双链断裂(double-strandedbreaks,DSBs),利用同源定向修复(homology-directedrepair,HDR)可实现对靶基因的特异性基因敲除(knock-out)或敲入(knock-in)。传统的技术方案将CRISPR/Cas9技术与Cre/loxP或FLP/FRT系统联用,可实现高效的基因打靶,也易于移除打靶过程中引入的筛选标记。然而,筛选标记移除过程中会在基因组中残留34个碱基的标签序列。因此,对基因组进行精确编辑的同时不引入无关序列仍有一定难度。在人工诱导多能干细胞(induced pluripotent stem cells, iPSCs)的基因组编辑中,CRISPR/Cas9技术和piggyBac转座酶联用的两步法策略能够实现这一目标:首先运用CRISPR/Cas9技术,利用同源定向修复原理引入基因突变及筛选标记,然后利用piggyBac转座酶将筛选标记精确移除。借鉴该方法的技术原理,本研究对果蝇(Drosophila melanogaster)CG4894基因进行了无缝编辑(seamless genome editing),成功将该基因第18外显子上第21位的酪氨酸(tyrosine,Y)突变为半胱氨酸(cysteine,C),且测序结果显示基因组中除设计位点之外并无其他外源序列残留。CRISPR/Cas9技术和piggyBac转座酶联用策略为果蝇基因组的精确编辑提供了更多选择。

The typeⅡCRISPR/Cas9(clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPRassociated protein 9)is an efficient RNA-guided genome-editing technique.Guided by sgRNA,the Cas9 endonuclease generates site-specific double-stranded breaks(DSB)at specific site,which is amenable to repair by homology-directed repair(HDR)to generate a designed knock-out or knock-in transgene.In combination with CRISPR/Cas9 and Cre/loxP or FLP/FRT system,efficient gene targeting can be achieved,and meanwhile screening markers introduced can be readily removed except a 34-base pair residual fragment.Thus,difficulties remain in accurate editing of the genome without introducing any extraneous sequences.In human induced pluripotent stem cells(iPSCs),a two-step strategy has been developed using CRISPR/Cas9 and the piggyBac system to establish a seamless genomic editing,in which CRISPR/Cas9 is initially used to introduce mutations along with screening markers by HDR,then the markers are precisely excised by piggyBac transposase.Using this strategy,we have successfully transformed the tyrosine to cysteine at position 21 within the 18th exon of the CG4894 gene in the Drosophila genome without introducing any extraneous sequence.Hence,this strategy provides more options for precise and seamless editing of the Drosophila genome.