CRISPR RNA Array-Guided Multisite Cleavage for Gene Disruption by Cas9 and Cpf1.

To achieve multisite-targeting-based DNA cleavage simultaneously, we designed two kinds of CRISPR RNA arrays by fusing four single guide RNAs (sgRNAs for Cas9 or crRNAs for Cpf1) with uncleavable RNA linkers (CRISPRay). The CRISPRay could operate on four adjacent target sites to cleave target DNA in a collaborative manner. Two CRISPR RNA arrays demonstrated robust inactivation of the firefly luciferase gene in living cells. In vitro DNA cleavage and DNA sequencing also verified that sgRNA arrays directed SpCas9 nuclease to cut target DNA at four cleavage sites simultaneously whereas crRNA-array-guided FnCpf1 nuclease showed target-activated, nonspecific DNase activity on both target DNA and nontarget DNA at random sites. Through optimization of the ratio of nuclease and CRIPSR RNAs, CRISPRay should further enhance gene interference in cells. This work presents a simple approach through which to improve multisite-directed gene disruption by fusing four guide RNAs (sgRNAs or crRNAs) into a CRISPR RNA string.