The RNA-guided endonuclease Cpf1 is a promising tool for genome editing in eukaryotic cells1–7. However, the utility of the commonly used Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1) and Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1) is limited by their requirement of a TTTV protospacer adjacent motif (PAM) in the DNA substrate. To address this limitation, we performed a structure-guided mutagenesis screen to increase the targeting range of Cpf1. We engineered two AsCpf1 variants carrying the mutations S542R/K607R and S542R/K548V/N552R, which recognize TYCV and TATV PAMs, respectively, with enhanced activities in vitro and in human cells. Genome-wide assessment of off-target activity using BLISS7 assay indicated that these variants retain high DNA targeting specificity, which we further improved by introducing an additional non-PAM-interacting mutation. Introducing the identified mutations at their corresponding positions in LbCpf1 similarly altered its PAM specificity. Together, these variants increase the targeting range of Cpf1 by approximately three-fold in human coding sequences to one cleavage site per ~11 bp.

Precisely measuring the location and frequency of DNA double-strand breaks (DSBs) along the genome is instrumental to understanding genomic fragility, but current methods are limited in versatility, sensitivity or practicality. Here we present Breaks Labeling In Situ and Sequencing (BLISS), featuring the following: (1) direct labelling of DSBs in fixed cells or tissue sections on a solid surface; (2) low-input requirement by linear amplification of tagged DSBs by in vitro transcription; (3) quantification of DSBs through unique molecular identifiers; and (4) easy scalability and multiplexing. We apply BLISS to profile endogenous and exogenous DSBs in low-input samples of cancer cells, embryonic stem cells and liver tissue. We demonstrate the sensitivity of BLISS by assessing the genome-wide off-target activity of two CRISPR-associated RNA-guided endonucleases, Cas9 and Cpf1, observing that Cpf1 has higher specificity than Cas9. Our results establish BLISS as a versatile, sensitive and efficient method for genome-wide DSB mapping in many applications.

Cyclic GMP (cGMP) signalling regulates multiple biological functions through activation of protein kinase G and cyclic nucleotide-gated (CNG) channels. In sensory neurons, cGMP permits signal modulation, amplification and encoding, before depolarization. Here we implement a guanylyl cyclase rhodopsin from Blastocladiella emersonii as a new optogenetic tool (BeCyclOp), enabling rapid light-triggered cGMP increase in heterologous cells (Xenopus oocytes, HEK293T cells) and in Caenorhabditis elegans. Among five different fungal CyclOps, exhibiting unusual eight transmembrane topologies and cytosolic N-termini, BeCyclOp is the superior optogenetic tool (light/dark activity ratio: 5,000; no cAMP production; turnover (20 °C) ∼17 cGMP s−1). Via co-expressed CNG channels (OLF in oocytes, TAX-2/4 in C. elegans muscle), BeCyclOp photoactivation induces a rapid conductance increase and depolarization at very low light intensities. In O2/CO2 sensory neurons of C. elegans, BeCyclOp activation evokes behavioural responses consistent with their normal sensory function. BeCyclOp therefore enables precise and rapid optogenetic manipulation of cGMP levels in cells and animals.

Human renal clear cell carcinoma (RCC) is frequently associated with loss of the von Hippel-Lindau (VHL)tumor suppressor (pVHL), which inhibits ubiquitylation and degradation of the alpha subunits of hypoxiainducible transcription factor. pVHL also ubiquitylates the large subunit of RNA polymerase II, Rpb1, phosphorylated on serine 5 (Ser5) within the C-terminal domain (CTD). A hydroxylated proline 1465 within an LXXLAP motif located N-terminal to the CTD allows the interaction of Rpb1 with pVHL. Here we report that in RCC cells, pVHL regulates expression of Rpb1 and is necessary for low-grade oxidative-stress-induced recruitment of Rpb1 to the DNA-engaged fraction and for its P1465 hydroxylation, phosphorylation, and nondegradative ubiquitylation. Egln-9-type prolyl hydroxylases, PHD1 and PHD2, coimmunoprecipitated with Rpb1 in the chromatin fraction of VHL ؉ RCC cells in response to oxidative stress, and PHD1 was necessary for P1465 hydroxylation while PHD2 had an inhibitory effect. P1465 hydroxylation was required for oxidativestress-induced Ser5 phosphorylation of Rpb1. Importantly, overexpression of wild-type Rpb1 stimulated formation of kidney tumors by VHL ؉ cells, and this effect was abolished by P1465A mutation of Rpb1. These data indicate that through this novel pathway involving P1465 hydroxylation and Ser5 phosphorylation of Rbp1, pVHL may regulate tumor growth.pVHL is the main tumor suppressor for which loss of activity is causatively linked to renal clear cell carcinoma (RCC), the most malignant and common form of kidney cancer. The VHL gene is mutated or hypermethylated in about 40 to 70% of sporadic RCC. Hereditary loss of pVHL function in von Hippel-Lindau (VHL) disease also results in highly vascularized RCC and capillary tumors of other organs, such as hemangioblastoma of the central nervous system and retinal angioma (19,20). A body of experimental evidence, based on a subcutaneous xenograft model system, supports the idea that accumulation of the alpha subunit of the hypoxia-inducible transcription factor (HIF) HIF-2␣ and induction of HIF target gene products, resulting from the loss of pVHL-mediated ubiquitylation, are necessary and sufficient to promote growth of RCC tumors (22,23). HIF activation has also been demonstrated as an early tumorigenesis event in kidneys from VHL patients (32). Biochemically, pVHL is the substrate-recognizing component of a multiprotein E3 ubiquitin ligase complex containing elongins C and B, Cullin 2, and the RING-H2 finger protein Rbx-1 (for a review, see reference 19). pVHL-dependent ubiquitylation of HIF-␣s is preceded by hydroxylation of conserved proline residues located within LXXLAP motifs (16,17) by the O 2 -, Fe(II)-, and oxyglutarate-regulated Egl-9-type proline hydroxylases (PHDs) (7). Thus, an important aspect of pVHL's tumor suppressing activity is the prevention of HIF-␣ accumulation, which in turn suppresses induction of the HIF target genes. Clearly, however, pVHL activity is not limited to regulation of HIFs. Other targets of pVHL-associated E...

Highlights d Few tools allow inhibition of neural activity for long time periods with light d We engineered botulinum neurotoxin B so that it can be activated with blue light d Photoactivated botulinum toxin efficiently cleaves the SNARE protein VAMP2 d We demonstrate utility in diverse systems, from mammalian brain slices to worms