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Over the last decade, research on distinct types of CRISPR systems has revealed many structural and functional variations. Recently, several novel types of single-polypeptide CRISPR-associated systems have been discovered including Cas12a/Cpf1 and Cas13a/C2c2. Despite distant similarities to Cas9, these additional systems have unique structural and functional features, providing new opportunities for genome editing applications. Here, relevant fundamental features of natural and engineered CRISPR-Cas variants are compared. Moreover, practical matters are discussed that are essential for dedicated genome editing applications, including nuclease regulation and delivery, target specificity, as well as host repair diversity.

Genome editing has recently made a revolutionary development with the introduction of the CRISPR–Cas technology. The programmable CRISPR-associated Cas9 and Cas12a nucleases generate specific dsDNA breaks in the genome, after which host DNA-repair mechanisms can be manipulated to implement the desired editing. Despite this spectacular progress, the efficiency of Cas9/Cas12a-based engineering can still be improved. Here, we address the variation in guide-dependent efficiency of Cas12a, and set out to reveal the molecular basis of this phenomenon. We established a sensitive and robust in vivo targeting assay based on loss of a target plasmid encoding the red fluorescent protein (mRFP). Our results suggest that folding of both the precursor guide (pre-crRNA) and the mature guide (crRNA) have a major influence on Cas12a activity. Especially, base pairing of the direct repeat, other than with itself, was found to be detrimental to the activity of Cas12a. Furthermore, we describe different approaches to minimize base-pairing interactions between the direct repeat and the variable part of the guide. We show that design of the 3′ end of the guide, which is not involved in target strand base pairing, may result in substantial improvement of the guide's targeting potential and hence of its genome editing efficiency.

Microbial CRISPR-Cas defense systems have been adapted as a platform for genome editing applications built around the RNA-guided effector nucleases, such as Cas9. We recently reported the characterization of Cpf1, the effector nuclease of a novel type V-A CRISPR system, and demonstrated that it can be adapted for genome editing in mammalian cells (Zetsche et al., 2015). Unlike Cas9, which utilizes a trans-activating crRNA (tracrRNA) as well as the endogenous RNaseIII for maturation of its dual crRNA:tracrRNA guides (Deltcheva et al., 2011), guide processing of the Cpf1 system proceeds in the absence of tracrRNA or other Cas (CRISPR associated) genes (Zetsche et al., 2015) (Figure 1a), suggesting that Cpf1 is sufficient for pre-crRNA maturation. This has important implications for genome editing, as it would provide a simple route to multiplex targeting. Here, we show for two Cpf1 orthologs that no other factors are required for array processing and demonstrate multiplex gene editing in mammalian cells as well as in the mouse brain by using a designed single CRISPR array.To confirm our previous observation that Cpf1 alone is sufficient for array processing (Zetsche et al., 2015), we synthesized an artificial CRISPR pre-crRNA array consisting of four spacers separated by direct repeats (DRs) from the CRISPR locus of Francisella novicida (FnCpf1) locus and tested the pre-crRNA processing activity in vitro of two Cpf1 orthologs with activity in mammalian cells, Acidaminococcus Cpf1 (AsCpf1) and Lachnospiraceae Cpf1 (LbCpf1). Both AsCpf1 and LbCpf1 cleaved the CRISPR array in similar patterns within 10 min, whereas they did not cleave a control RNA lacking DR sequence features (Figure 1b). These findings are consistent with a recent report that FnCpf1 is the only required component for crRNA processing in vitro and in heterologous systems (Fonfara et al., 2016). The typical cleavage pattern suggests that Cpf1 recognizes secondary structures and/or motifs on its pre-crRNA array, which is also in agreement with observations from FnCpf1 (Fonfara et al., 2016). Moreover, it reinforces our previous finding that the DR sequences of Cpf1 family proteins are highly conserved and functionally interchangeable (Zetsche et al., 2015). We analyzed an array cleaved with AsCpf1 by small RNAseq and confirmed that the cleavage products correlate to fragments resulting from cuts at the 5' end of each DR hairpin, identical to the cleavage pattern we observed previously in E.coli heterologously expressing FnCpf1 CRISPR systems (Zetsche et al., 2015) (Figure 1c). 3We further validated these results by generating Cpf1 mutants that are unable to process arrays.Guided by the crystal structure of AsCpf1 (Yamano et al., 2016), we identified five conserved amino acid residues close to the 5' end of the DR that are likely to disrupt array processing and mutated them to alanine (H800, K809, K860, F864, and R790) (Yamano et al., 2016). All of these mutations interfered with pre-crRNA processing but not with DNA cleavage activity in vitro (Figure 2...

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