Programmable downsizing of CRISPR-Cas9 activity for precise and safe genome editing

Abstract

CRSIPR-Cas9 system has opened up the avenue to efficient genome editing1-4. However, together with known off-target effects, several concerns of current CRISPR-Cas9 platform, including severe DNA damage, cytotoxicity, and large genomic alteration, have emerged in recent reports5-7 and establish a formidable obstacle to precisely model allele dosage effects of disease mutations and risk variants, especially mono-allelic effects, and correct them. Here, by developing an allele-specific indel monitor system (AIMS), we demonstrate that small and simple modification of conventional single-guide RNAs (sgRNAs) enable programmable tuning of CRISPR-Cas9 activities and alleviate such adverse effects. AIMS, which visualizes various indel events in two alleles separately in living cells, is convenient and accurate to determine the in vitro editing efficiency and revealed frequent mosaicism during genome editing. Using AIMS, we show that adding cytosine stretches to the 5 end of conventional sgRNA efficiently reduced Cas9 activity in a length dependent manner. By combining systematic experiments and computational modeling, we established the quantitative relationships between the length of cytosine extension and multiple aspects of CRISPR-Cas9 system. In general, short cytosine extension dramatically relieves p53-dependent cytotoxicity and suppression of homology-directed repair (HDR) while relatively maintaining on-target activity. Long cytosine extension further decreases on-target activity, thereby maximizing mono-allelic editing, while conventional system typically induces bi-allelic editing. Furthermore, such downregulation of on-target activity contributes to downregulation of relative off-target activity and protection of HDR-allele from second off-target editing. Therefore, cytosine extension method finally enables both single-step generation of heterozygous single-nucleotide disease mutations from homozygous states in mouse ES cells and correction of heterozygous disease mutations in human iPS cells. Taken together, our study proposes updates of standard CRISPR-Cas9 platform in mammalian cells toward precise and safe genome editing in diverse applications.