包含Cas核酸酶和指导RNA(gRNA)的CRISPR技术可以利用多个gRNA在同一细胞中进行多位点编辑或调控。Nature设计了一种高度紧凑的方法,以CRISPR阵列的形式编码gRNA,该阵列由通过靶向间隔区隔开的保守重复序列组成。然而,获得新间隔物的能力使阵列比CRISPR技术所需的时间更长。这里,我们表明,Cas9核酸酶使用的CRISPR阵列可以缩短而不损害,有时甚至增强靶向活性。利用大肠杆菌中的多重基因抑制,我们发现,在严重损害靶向活性之前,每个区域都可以被系统性地缩短至不同程度.令人惊讶的是,缩短一些间隔区产生增强的靶向活性,这与处理前转录阵列的折叠有关。总的来说,缩短的CRISPR-Cas9阵列可以促进在CRISPR技术的许多细菌应用中从较小的DNA足迹进行多重编辑和基因调控。
CRISPR technologies comprising a Cas nuclease and a guide RNA (gRNA) can utilize multiple gRNAs to enact multi-site editing or regulation in the same cell. Nature devised a highly compact means of encoding gRNAs in the form of CRISPR arrays composed of conserved repeats separated by targeting spacers. However, the capacity to acquire new spacers keeps the arrays longer than necessary for CRISPR technologies. Here, we show that CRISPR arrays utilized by the Cas9 nuclease can be shortened without compromising and sometimes even enhancing targeting activity. Using multiplexed gene repression in E. coli, we found that each region could be systematically shortened to varying degrees before severely compromising targeting activity. Surprisingly, shortening some spacers yielded enhanced targeting activity, which was linked to folding of the transcribed array prior to processing. Overall, shortened CRISPR-Cas9 arrays can facilitate multiplexed editing and gene regulation from a smaller DNA footprint across many bacterial applications of CRISPR technologies.