No abstract

CRISPR/Cas12a is a single effector nuclease that, like CRISPR/Cas9, has been harnessed for genome editing based on its ability to generate targeted DNA double strand breaks (DSBs). Unlike the blunt-ended DSB generated by Cas9, Cas12a generates sticky-ended DSB that could potentially aid precise genome editing, but this unique feature has thus far been underutilized. In the current study, we found that a short double-stranded DNA (dsDNA) repair template containing a sticky end that matched one of the Cas12a-generated DSB ends and a homologous arm sharing homology with the genomic region adjacent to the other end of the DSB enabled precise repair of the DSB and introduced a desired nucleotide substitution. We termed this strategy ‘Ligation-Assisted Homologous Recombination’ (LAHR). Compared to the single-stranded oligo deoxyribonucleotide (ssODN)-mediated homology directed repair (HDR), LAHR yields relatively high editing efficiency as demonstrated for both a reporter gene and endogenous genes. We found that both HDR and microhomology-mediated end joining (MMEJ) mechanisms are involved in the LAHR process. Our LAHR genome editing strategy, extends the repertoire of genome editing technologies and provides a broader understanding of the type and role of DNA repair mechanisms involved in genome editing.

CRISPR-Cas13 systems are unique among Class II CRISPR systems, as they exclusively target RNA. In vitro and in prokaryotic cells, Cas13 cleaves both target and non-target RNA indiscriminately upon activation by a specific target RNA. This property has been exploited for development of diagnostic nucleic acid detection tools. In eukaryotic cells, CRISPR-Cas13 initially seemed to exclusively cleave the target RNA and consequently, CRISPR-Cas13 has been adopted as a specific RNA knockdown tool. Recently, several groups have reported unexpected toxicity or collateral cleavage when using CRISPR-Cas13 in eukaryotic cells, which seems difficult to reconcile with the reported target specificity. To understand these seemingly contradicting findings, we explored the collateral cleavage activity of six Cas13 systems, and show that only the most active ortholog in vitro, LbuCas13a, exhibits strong collateral RNA cleavage activity in human cells. LbuCas13a displayed collateral cleavage in all tested cell lines, targeting both exogenous and endogenous transcripts and using different RNP delivery methods. Using Nanopore sequencing, we found that cytoplasmic RNAs are cleaved without bias by LbuCas13a. Furthermore, the cleavage sites are highly specific and often present in Uracil containing single stranded RNA loops of stem-loop structures. In response to collateral RNA cleavage, cells upregulate stress and innate immune response genes and depending on target transcript levels, RNA degradation resulted in apoptotic cell death. We demonstrate that LbuCas13a can serve as a cell selection tool, killing cells in a target RNA specific manner. As such, CRISPR-Cas13 is a promising new technology that might be useful in anti-tumor applications.