Genome-wide specificity profiles of CRISPR-Cas Cpf1 nucleases in human cells

The kinetic basis of CRISPR-Cas off-targeting rules

“…This versatility, together with the ability to design synthetic gRNA complementary to any target of choice, holds great promise for gene editing and gene silencing applications (1)(2)(3)(4). Among the known RGNs, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated (Cas) nucleases Cas9 (3,(5)(6)(7)(8) and Cpf1 (Cas12a (9)) (10)(11)(12) are of special interest, as they are comparatively simple single-subunit enzymes. Cas nucleases have been successfully used to target sequences in genomes from a variety of organisms, ranging from prokaryotes to mammalian cells (7,(11)(12)(13)(14)(15).…”

“…Among the known RGNs, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated (Cas) nucleases Cas9 (3,(5)(6)(7)(8) and Cpf1 (Cas12a (9)) (10)(11)(12) are of special interest, as they are comparatively simple single-subunit enzymes. Cas nucleases have been successfully used to target sequences in genomes from a variety of organisms, ranging from prokaryotes to mammalian cells (7,(11)(12)(13)(14)(15). Recent success stories include the correction of point mutations relevant to human disease (16), the construction of sequence specific antimicrobials (17), the conveyance of infertility in malaria carrying mosquitoes (18), and the inactivation of retroviral elements to facilitate porcine-to-human organ transplants (19).…”

“…As viruses evolve in response to the selective pressure induced by the CRISPR immune system, the host is in turn under pressure to not only attack the target, but to also attack slightly mutated target sequences (24)(25)(26). It is therefore not surprising that Cas nucleases exhibit considerable off-target activity on sequences similar to the intended target (11,12,15,(27)(28)(29)(30)(31)(32)(33)(34). Such off-target activity presents a severe problem for any therapeutic applications (4), as DNA breaks introduced at the wrong site could lead to loss-of-function mutations in a wellfunctioning gene, or the improper repair of a disease causing gene.…”

“…Surprisingly, the level of complementarity to the guide alone does not determine how strongly a particular sequence is targeted. To shed light on the determinants of off-target activity, a recent flurry of experiments has systematically probed the level of binding and/or cleavage on mutated target sequences (3), including high-throughput screens of large libraries of off-targets (11,12,14,27,(29)(30)(31)(33)(34)(35)(36), biochemical studies (6,8,10,35,37,38), structural studies (39)(40)(41)(42)(43)(44)(45), and single-molecule biophysical studies (46)(47)(48)(49)(50)(51)(52)(53) providing insights into the mechanics of targeting. To date, a number of rather peculiar targeting rules have been established for Cas nucleases: (i) specificity-efficiency decoupling: weakened protein-DNA interactions can improve target selectivity while still maintaining efficiency (54)(55)(56)(57); (ii) seed region: single mismatches within a so-called seed region (a stretch of nucleotides following the PAM (58)) can completely disrupt interference, while mismatches further into the guide have much less of an effect (6, 8, 11, 12, 15, 27-34, 38, 47, 48, 50); (iii) mismatch spread: when mismatches are outside the seed region, off-targets with spread out mismatches are targeted most strongly (27,…”

“…Though we frame our considerations in terms of the well-studied and technologically important Cas9, our approach applies to any RGN that displays a progressive matching between guide and targeted (40,47,48,50,51) before cleavage (41,72) (Figure 1A). To demonstrate the generality and power of our approach, we present fits to targeting data from Argonaute 2 (hAgo2), as well as type I, II and V CRISPR systems (11,31,38,52).…”