Enzyme Kinetics and Detector Sensitivity Determine Limits of Detection of Amplification-Free CRISPR-Cas12 and CRISPR-Cas13 Diagnostics.

Interest in CRISPR-Cas12 and CRISPR-Cas13 detection continues to increase as these detection schemes enable the specific recognition of nucleic acids. The fundamental sensitivity limits of these schemes (and their applicability in amplification-free assays) are governed by kinetic rates. However, these kinetic rates remain poorly understood, and their reporting has been inconsistent. We quantify kinetic parameters for several enzymes (LbCas12a, AsCas12a, AapCas12b, LwaCas13a, and LbuCas13a) and their corresponding limits of detection (LoD). Collectively, we present quantification of enzyme kinetics for 14 guide RNAs (gRNAs) and nucleic acid targets for a total of 50 sets of kinetic rate parameters and 25 LoDs. We validate the self-consistency of our measurements by comparing trends and limiting behaviors with a Michaelis-Menten trans-cleavage reaction kinetics model. For our assay conditions, activated Cas12 and Cas13 enzymes exhibit trans-cleavage catalytic efficiencies between order 105 and 106 M-1 s-1. For assays that use fluorescent reporter molecules (ssDNA and ssRNA) for target detection, the kinetic rates at the current assay conditions result in an amplification-free LoD in the picomolar range. The results suggest that successful detection of target requires cleavage (by an activated CRISPR enzyme) of the order of at least 0.1% of the fluorescent reporter molecules. This fraction of reporters cleaved is required to differentiate the signal from the background, and we hypothesize that this required fraction is largely independent of the detection method (e.g., endpoint vs reaction velocity) and detector sensitivity. Our results demonstrate the fundamental nature by which kinetic rates and background signal limit LoDs and thus highlight areas of improvement for the emerging field of CRISPR diagnostics.

RNA-Seq analysis to capture the transcriptome landscape of a single cell.

We describe here a protocol for digital transcriptome analysis in a single mouse oocyte and blastomere using a deep-sequencing approach. In this method, individual cells are isolated and transferred into lysate buffer by mouth pipette, followed by reverse transcription carried out directly on the whole cell lysate. Free primers are removed by exonuclease I and a poly(A) tail is added to the 3' end of the first-strand cDNAs by terminal deoxynucleotidyl transferase. Single-cell cDNAs are then amplified by 20 + 9 cycles of PCR. The resulting 100-200 ng of amplified cDNAs are used to construct a sequencing library, which can be used for deep sequencing using the SOLiD system. Compared with cDNA microarray techniques, our assay can capture up to 75% more genes expressed in early embryos. This protocol can generate deep-sequencing libraries for 16 single-cell samples within 6 d.

A truncated DNA-damage-signaling response is activated after DSB formation in the G1 phase of Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the DNA damage response (DDR) is activated by the spatio-temporal colocalization of Mec1-Ddc2 kinase and the 9-1-1 clamp. In the absence of direct means to monitor Mec1 kinase activation in vivo, activation of the checkpoint kinase Rad53 has been taken as a proxy for DDR activation. Here, we identify serine 378 of the Rad55 recombination protein as a direct target site of Mec1. Rad55-S378 phosphorylation leads to an electrophoretic mobility shift of the protein and acts as a sentinel for Mec1 activation in vivo. A single double-stranded break (DSB) in G1-arrested cells causes phosphorylation of Rad55-S378, indicating activation of Mec1 kinase. However, Rad53 kinase is not detectably activated under these conditions. This response required Mec1-Ddc2 and loading of the 9-1-1 clamp by Rad24-RFC, but not Rad9 or Mrc1. In addition to Rad55-S378, two additional direct Mec1 kinase targets are phosphorylated, the middle subunit of the ssDNA-binding protein RPA, RPA2 and histone H2A (H2AX). These data suggest the existence of a truncated signaling pathway in response to a single DSB in G1-arrested cells that activates Mec1 without eliciting a full DDR involving the entire signaling pathway including the effector kinases.

Genetic analysis reveals different roles of Schizosaccharomyces pombe sfr1/dds20 in meiotic and mitotic DNA recombination and repair.

DNA double-strand break (DSB) repair mediated by the Rad51 pathway of homologous recombination is conserved in eukaryotes. In yeast, Rad51 paralogs, Saccharomyces cerevisiae Rad55-Rad57 and Schizosaccharomyces pombe Rhp55-Rhp57, are mediators of Rad51 nucleoprotein formation. The recently discovered S. pombe Sfr1/Dds20 protein has been shown to interact with Rad51 and to operate in the Rad51-dependent DSB repair pathway in parallel to the paralog-mediated pathway. Here we show that Sfr1 is a nuclear protein and acts downstream of Rad50 in DSB processing. sfr1Delta is epistatic to rad18 (-) and rad60 (-), and Sfr1 is a high-copy suppressor of the replication and repair defects of a rad60 mutant. Sfr1 functions in a Cds1-independent UV damage tolerance mechanism. In contrast to mitotic recombination, meiotic recombination is significantly reduced in sfr1Delta strains. Our data indicate that Sfr1 acts in DSB repair mainly outside of S-phase, and is required for wild-type levels of meiotic recombination. We suggest that Sfr1 acts early in recombination and has a specific role in Rad51 filament assembly, distinct from that of the Rad51 paralogs.

Phosphorylation of Rad55 on serines 2, 8, and 14 is required for efficient homologous recombination in the recovery of stalled replication forks.

DNA damage checkpoints coordinate the cellular response to genotoxic stress and arrest the cell cycle in response to DNA damage and replication fork stalling. Homologous recombination is a ubiquitous pathway for the repair of DNA double-stranded breaks and other checkpoint-inducing lesions. Moreover, homologous recombination is involved in postreplicative tolerance of DNA damage and the recovery of DNA replication after replication fork stalling. Here, we show that the phosphorylation on serines 2, 8, and 14 (S2,8,14) of the Rad55 protein is specifically required for survival as well as for normal growth under genome-wide genotoxic stress. Rad55 is a Rad51 paralog in Saccharomyces cerevisiae and functions in the assembly of the Rad51 filament, a central intermediate in recombinational DNA repair. Phosphorylation-defective rad55-S2,8,14A mutants display a very slow traversal of S phase under DNA-damaging conditions, which is likely due to the slower recovery of stalled replication forks or the slower repair of replication-associated DNA damage. These results suggest that Rad55-S2,8,14 phosphorylation activates recombinational repair, allowing for faster recovery after genotoxic stress.

DNA damage-induced phosphorylation of Rad55 protein as a sentinel for DNA damage checkpoint activation in S. cerevisiae.

Rad55 protein is one of two Rad51 paralogs in the budding yeast Saccharomyces cerevisiae and forms a stable heterodimer with Rad57, the other Rad51 paralog. The Rad55-Rad57 heterodimer functions in homologous recombination during the assembly of the Rad51-ssDNA filament, which is central for homology search and DNA strand exchange. Previously, we identified Rad55 protein as a terminal target of the DNA damage checkpoints, which coordinate the cellular response to genotoxic stress. Rad55 protein phosphorylation is signaled by a significant electrophoretic shift and occurs in response to a wide range of genotoxic stress. Here, we map the phosphorylation site leading to the electrophoretic shift and show that Rad55 protein is a bona fide direct in vivo substrate of the central DNA damage checkpoint kinase Mec1, the budding yeast equivalent of human ATM/ATR. We provide protocols to monitor the Rad55 phosphorylation status in vivo and assay Rad55-Rad57 phosphorylation in vitro using purified substrate with the Mec1 and Rad53 checkpoint kinases.

Esc4/Rtt107 and the control of recombination during replication.

When replication forks stall during DNA synthesis, cells respond by assembling multi-protein complexes to control the various pathways that stabilize the replication machinery, repair the replication fork, and facilitate the reinitiation of processive DNA synthesis. Increasing evidence suggests that cells have evolved scaffolding proteins to orchestrate and control the assembly of these repair complexes, typified in mammalian cells by several BRCT-motif containing proteins, such as Brca1, Xrcc1, and 53BP1. In Saccharomyces cerevisiae, Esc4 contains six such BRCT domains and is required for the most efficient response to a variety of agents that damage DNA. We show that Esc4 interacts with several proteins involved in the repair and processing of stalled or collapsed replication forks, including the recombination protein Rad55. However, the function of Esc4 does not appear to be restricted to a Rad55-dependent process, as we observed an increase in sensitivity to the DNA alkylating agent methane methylsulfonate (MMS) in a esc4Deltarad55Delta mutant, as well as in double mutants of esc4Delta and other recombination genes, compared to the corresponding single mutants. In addition, we show that Esc4 forms multiple nuclear foci in response to treatment with MMS. Similar behavior is also observed in the absence of damage when either of the S-phase checkpoint proteins, Tof1 or Mrc1, is deleted. Thus, we propose that Esc4 associates with ssDNA of stalled forks and acts as a scaffolding protein to recruit and/or modulate the function of other proteins required to reinitiate DNA synthesis.

Identification and characterization of the rlp1+, the novel Rad51 paralog in the fission yeast Schizosaccharomyces pombe.

A new DNA repair gene from fission yeast Schizosaccharomyces pombe rlp1+ (RecA-like protein) has been identified. Rlp1 shows homology to RecA-like proteins, and is the third S. pombe Rad51 paralog besides Rhp55 and Rhp57. The new gene encodes a 363 aa protein with predicted Mr of 41,700 and has NTP-binding motif. The rlp1Delta mutant is sensitive to methyl methanesulfonate (MMS), ionizing radiation (IR), and camptothecin (CPT), although to a lesser extent than the deletion mutants of rhp55+ and rhp51+ genes. In contrast to other recombinational repair mutants, the rlp1Delta mutant does not exhibit sensitivity to UV light and mitomycin C (MMC). Mitotic recombination is moderately reduced in rlp1 mutant. Epistatic analysis of MMS and IR-sensitivity of rlp1Delta mutant indicates that rlp1+ acts in the recombinational pathway of double-strand break (DSB) repair together with rhp51+, rhp55+, and rad22+ genes. Yeast two-hybrid analysis suggests that Rlp1 may interact with Rhp57 protein. We propose that Rlp1 have an accessory role in repair of a subset of DNA damage induced by MMS and IR, and is required for the full extent of DNA recombination and cell survival under condition of a replication fork collapse.

Amino acid changes in Xrs2p, Dun1p, and Rfa2p that remove the preferred targets of the ATM family of protein kinases do not affect DNA repair or telomere length in Saccharomyces cerevisiae.

In eukaryotes, mutations in a number of genes that affect DNA damage checkpoints or DNA replication also affect telomere length [Curr. Opin. Cell Biol. 13 (2001) 281]. Saccharomyces cerevisae strains with mutations in the TEL1 gene (encoding an ATM-like protein kinase) have very short telomeres, as do strains with mutations in XRS2, RAD50, or MRE11 (encoding members of a trimeric complex). Xrs2p and Mre11p are phosphorylated in a Tel1p-dependent manner in response to DNA damage [Genes Dev. 15 (2001) 2238; Mol. Cell 7 (2001) 1255]. We found that Xrs2p, but not Mre11p or Rad50p, is efficiently phosphorylated in vitro by immunopreciptated Tel1p. Strains with mutations eliminating all SQ and TQ motifs in Xrs2p (preferred targets of the ATM kinase family) had wild-type length telomeres and wild-type sensitivity to DNA damaging agents. We also showed that Rfa2p (a subunit of RPA) and the Dun1p checkpoint kinase, which are required for DNA damage repair and which are phosphorylated in response to DNA damage in vivo, are in vitro substrates of the Tel1p and Mec1p kinases. In addition, Dun1p substrates with no SQ or TQ motifs are phosphorylated by Mec1p in vitro very inefficiently, but retain most of their ability to be phosphorylated by Tel1p. We demonstrated that null alleles of DUN1 and certain mutant alleles of RFA2 result in short telomeres. As observed with Xrs2p, however, strains with mutations of DUN1 or RFA2 that eliminate SQ motifs have no effect on telomere length or DNA damage sensitivity.

Direct kinase-to-kinase signaling mediated by the FHA phosphoprotein recognition domain of the Dun1 DNA damage checkpoint kinase.

The serine-threonine kinase Dun1 contains a forkhead-associated (FHA) domain and functions in the DNA damage checkpoint pathway of Saccharomyces cerevisiae. It belongs to the Chk2 family of checkpoint kinases, which includes S. cerevisiae Rad53 and Mek1, Schizosaccharomyces pombe Cds1, and human Chk2. Dun1 is required for DNA damage-induced transcription of certain target genes, transient G(2)/M arrest after DNA damage, and DNA damage-induced phosphorylation of the DNA repair protein Rad55. Here we report that the FHA phosphoprotein recognition domain of Dun1 is required for direct phosphorylation of Dun1 by Rad53 kinase in vitro and in vivo. trans phosphorylation by Rad53 does not require the Dun1 kinase activity and is likely to involve only a transient interaction between the two kinases. The checkpoint functions of Dun1 kinase in DNA damage-induced transcription, G(2)/M cell cycle arrest, and Rad55 phosphorylation are severely compromised in an FHA domain mutant of Dun1. As a consequence, the Dun1 FHA domain mutant displays enhanced sensitivity to genotoxic stress induced by UV, methyl methanesulfonate, and the replication inhibitor hydroxyurea. We show that the Dun1 FHA domain is critical for direct kinase-to-kinase signaling from Rad53 to Dun1 in the DNA damage checkpoint pathway.