CRISPR-Cas nucleases are powerful tools for manipulating nucleic acids; however, targeted insertion of DNA remains a challenge, as it requires host cell repair machinery. Here we characterize a CRISPR-associated transposase from cyanobacteria Scytonema hofmanni (ShCAST) that consists of Tn7-like transposase subunits and the type V-K CRISPR effector (Cas12k). ShCAST catalyzes RNA-guided DNA transposition by unidirectionally inserting segments of DNA 60 to 66 base pairs downstream of the protospacer. ShCAST integrates DNA into targeted sites in the Escherichia coli genome with frequencies of up to 80% without positive selection. This work expands our understanding of the functional diversity of CRISPR-Cas systems and establishes a paradigm for precision DNA insertion.

Mitotic cells inactivate DNA double-strand break (DSB) repair, but the rationale behind this suppression remains unknown. Here, we unravel how mitosis blocks DSB repair and determine the consequences of repair reactivation. Mitotic kinases phosphorylate the E3 ubiquitin ligase RNF8 and the nonhomologous end joining factor 53BP1 to inhibit their recruitment to DSB-flanking chromatin. Restoration of RNF8 and 53BP1 accumulation at mitotic DSB sites activates DNA repair but is, paradoxically, deleterious. Aberrantly controlled mitotic DSB repair leads to Aurora B kinase-dependent sister telomere fusions that produce dicentric chromosomes and aneuploidy, especially in the presence of exogenous genotoxic stress. We conclude that the capacity of mitotic DSB repair to destabilize the genome explains the necessity for its suppression during mitosis, principally due to the fusogenic potential of mitotic telomeres.

No abstract

Highlights d Tagmentation-based tag integration site sequencing (TTISS) scalably detects DSBs d TTISS is a rapid and streamlined protocol compatible with multiplexing d Application of TTISS highlights trade-off in Cas9 variant specificity and activity d LZ3 Cas9 variant exhibits a unique +1 insertion profile

The type-V CRISPR effector Cas12b (formerly known as C2c1) has been challenging to develop for genome editing in human cells, at least in part due to the high temperature requirement of the characterized family members. Here we explore the diversity of the Cas12b family and identify a promising candidate for human gene editing from Bacillus hisashii, BhCas12b. However, at 37 °C, wild-type BhCas12b preferentially nicks the non-target DNA strand instead of forming a double strand break, leading to lower editing efficiency. Using a combination of approaches, we identify gain-of-function mutations for BhCas12b that overcome this limitation. Mutant BhCas12b facilitates robust genome editing in human cell lines and ex vivo in primary human T cells, and exhibits greater specificity compared to S. pyogenes Cas9. This work establishes a third RNA-guided nuclease platform, in addition to Cas9 and Cpf1/Cas12a, for genome editing in human cells.

In budding yeast, chromatin mobility increases after a DNA double-strand break (DSB). This increase is dependent on Mec1, the yeast ATR kinase, but the targets responsible for this phenomenon are unknown. Here we report that the Mec1-dependent phosphorylation of Cep3, a kinetochore component, is required to stimulate chromatin mobility after DNA breaks. Cep3 phosphorylation counteracts a constraint on chromosome movement imposed by the attachment of centromeres to the spindle pole body. A second constraint, imposed by the tethering of telomeres to the nuclear periphery, is also relieved after chromosome breakage. A non-phosphorylatable Cep3 mutant that impairs DSB-induced chromatin mobility is proficient in DSB repair, suggesting that break-induced chromatin mobility may be dispensable for homology search. Rather, we propose that the relief of centromeric constraint promotes cell cycle arrest and faithful chromosome segregation through the engagement of the spindle assembly checkpoint.

The universally conserved Kae1/Qri7/YgjD and Sua5/YrdC protein families have been implicated in growth, telomere homeostasis, transcription and the N6-threonylcarbamoylation (t6A) of tRNA, an essential modification required for translational fidelity by the ribosome. In bacteria, YgjD orthologues operate in concert with the bacterial-specific proteins YeaZ and YjeE, whereas in archaeal and eukaryotic systems, Kae1 operates as part of a larger macromolecular assembly called KEOPS with Bud32, Cgi121, Gon7 and Pcc1 subunits. Qri7 orthologues function in the mitochondria and may represent the most primitive member of the Kae1/Qri7/YgjD protein family. In accordance with previous findings, we confirm that Qri7 complements Kae1 function and uncover that Qri7 complements the function of all KEOPS subunits in growth, t6A biosynthesis and, to a partial degree, telomere maintenance. These observations suggest that Kae1 provides a core essential function that other subunits within KEOPS have evolved to support. Consistent with this inference, Qri7 alone is sufficient for t6A biosynthesis with Sua5 in vitro. In addition, the 2.9 Å crystal structure of Qri7 reveals a simple homodimer arrangement that is supplanted by the heterodimerization of YgjD with YeaZ in bacteria and heterodimerization of Kae1 with Pcc1 in KEOPS. The partial complementation of telomere maintenance by Qri7 hints that KEOPS has evolved novel functions in higher organisms.

SummaryThe recently discovered class 2 CRISPR-Cas endonuclease Cpf1 offers several advantages over Cas9, including the ability to process its own array and requirement for just a single RNA guide.These attributes make Cpf1 promising for many genome engineering applications. To further expand the suite of Cpf1 tools available, we tested 16 Cpf1 orthologs for activity in eukaryotic cells. Four of these new enzymes demonstrated targeted activity, one of which, from Moraxella bovoculi AAX11_00205 (Mb3Cpf1), exhibited robust indel formation. We also show that Mb3Cpf1 displays some tolerance for a shortened PAM (TTN versus the canonical Cpf1 PAM TTTV). The addition of these enzymes to the genome editing toolbox will further expand the utility of this powerful technology. * * * * * Class 2 CRISPR-Cas systems are naturally occurring microbial adaptive immune systems with single effector enzymes. The effector enzymes, such as Cas9, are RNA guided DNA endonucleases, which have been harnessed for a range of genome engineering applications (Doudna and Charpentier, 2014; Hsu et al., 2014). Although Cas9 was the first such enzyme to be developed as a genome editing tool (Cong et al., 2013; Mali et al., 2013), three orthologs of Cpf1, a single RNA-guided class 2 effector, from Francisella novicida U112 (FnCpf1), Acidaminococcus sp. BV3L6 (AsCpf1), and Lachnospiraceae bacterium ND2006 (LbCpf1), have also been used for genome editing in eukaryotic cells (Endo et al., 2016; Kim et al., 2016; Ma et al., 2017; Zetsche et al., 2015; Zetsche et al., 2016). Endonucleases of the Cpf1-family differ from the Cas9-family in several ways: (i) Cpf1 utilizes T-rich protospacer adjacent motifs (PAMs) located 5' of the targeted DNA sequence, (ii) target cleavage occurs distally from the PAM and results in sticky-end overhangs, (iii) Cpf1 is guided by a single CRISPR RNA (crRNA) and does not require trans-activating CRISPR RNA (tracrRNA); and (iv) Cpf1 possesses both RNase and DNase activity, which allows it to process its own CRISPR array (Fonfara et al., 2016; Zetsche et al., 2015). These features make Cpf1 particularly useful in certain situations, such as targeting AT-rich genomic regions and multiplexed gene targeting (Wang et al., 2017; Zetsche et al., 2016).. CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/134015 doi: bioRxiv preprint first posted online May. 4, 2017; 3 Given previous work showing that different Cas9 orthologs exhibit a range of activity in eukaryotic cells (Cong et al., 2013; Ran et al., 2015) and the potential advantages of Cpf1, we sought to identify additional Cpf1 orthologs with high activity in eukaryotic cells. Here we examine 16 new Cpf1-family proteins for nuclease activity in human cells. We identify four orthologs that can induce insertion/deletion (indel) events at targeted genomic loci. One ortholog, from Moraxella bovoculi AAX11_00205 (Mb3Cpf1), exhibited compara...