The repair outcomes at site-specific DNA double-strand breaks (DSBs) generated by the RNA-guided DNA endonuclease Cas9 determine how gene function is altered. Despite the widespread adoption of CRISPR-Cas9 technology to induce DSBs for genome engineering, the resulting repair products have not been examined in depth. Here, the DNA repair profiles of 223 sites in the human genome demonstrate that the pattern of DNA repair following Cas9 cutting at each site is nonrandom and consistent across experimental replicates, cell lines, and reagent delivery methods. Furthermore, the repair outcomes are determined by the protospacer sequence rather than genomic context, indicating that DNA repair profiling in cell lines can be used to anticipate repair outcomes in primary cells. Chemical inhibition of DNA-PK enabled dissection of the DNA repair profiles into contributions from c-NHEJ and MMEJ. Finally, this work elucidates a strategy for using "error-prone" DNA-repair machinery to generate precise edits.
RNA-guided CRISPR-Cas9 endonucleases are widely used for genome engineering, but our understanding of Cas9 specificity remains incomplete. Here, we developed a biochemical method (SITE-Seq), using Cas9 programmed with single-guide RNAs (sgRNAs), to identify the sequence of cut sites within genomic DNA. Cells edited with the same Cas9-sgRNA complexes are then assayed for mutations at each cut site using amplicon sequencing. We used SITE-Seq to examine Cas9 specificity with sgRNAs targeting the human genome. The number of sites identified depended on sgRNA sequence and nuclease concentration. Sites identified at lower concentrations showed a higher propensity for off-target mutations in cells. The list of off-target sites showing activity in cells was influenced by sgRNP delivery, cell type and duration of exposure to the nuclease. Collectively, our results underscore the utility of combining comprehensive biochemical identification of off-target sites with independent cell-based measurements of activity at those sites when assessing nuclease activity and specificity.
One Hundred Fifty-Seven nm photodissociation of singly protonated peptides generates unusual distributions of fragment ions. When the charge is localized at the C-terminus of the peptide, spectra are dominated by x-, v-, and w-type fragments. When it is sequestered at the N-terminus, a-and d-type ions are overwhelmingly abundant. Evidence is presented suggesting that the fragmentation occurs via photolytic radical cleavage of the peptide backbone at the bond between the ␣-and carbonyl-carbons followed by radical elimination to form the observed daughter ions. Low-energy fragmentation appears to be well described by the mobile proton model according to which vibrational excitation of the analyte leads to charge mobility [12]. Transfer of the charge proton to either the backbone carbonyl oxygen or amide nitrogen enables charge-induced cleavage of the peptide backbone. This process yields primarily b-and y-type fragment ions according to the standard nomenclature shown below [13,14].Higher energy activation methods involving collisions with gas molecules or surfaces can enable chargeremote fragmentation [15][16][17]. However, even in these cases, apparently, protonated peptides still generate some fragments through charge-directed processes [17]. Immobilization of the charge, either by using metal adducts [18] or charged chemical modifications [19 -22], has been used to inhibit charge-directed fragmentation. In these cases, primarily a-, d-, and w-type fragments are observed. Several mechanisms have been proposed for charge-remote peptide fragmentation, some involving homolytic radical cleavage [14,23].Electron capture dissociation (ECD) [24] and the recently reported electron-transfer dissociation (ETD)[25] appear to be nonthermal processes. These phenomena generate c-and z-type fragment ions upon the addition of an electron to a multiply charged protein or peptide ion. Two mechanisms have been proposed, one involving reactions of a free hydrogen atom generated when an electron is captured by a charged site on the analyte ion [26], and the other involving localization of the ϳ6 eV of energy that is generated upon charge neutralization. This energy then induces fragmentation through an excited electronic state [27]. Implicit in this second mechanism is the suggestion that techniques which excite appropriate electronic states can lead to unique, nonergodic fragmentation even for molecules as large as peptides and proteins [24,27]. This can occur if ions reach a dissociative electronic state and fragment before the energy is redistributed throughout the molecule. Electronic to vibrational relaxation, resulting in nonspecific excitation is an important competing process that often inhibits nonergodic processes, and rates of relaxation are predicted to increase with the size of the molecule [28].Lasers are powerful tools that can also be used to excite molecules to specific energy levels with either multiphoton or single photon processes. Light is not affected by the electric or magnetic fields of mass spectrometers, s...
Bond-selective chemistry has been a goal of photochemists for decades, particularly since the development and proliferation of tunable laser light sources. Nevertheless, for relatively large molecules, this goal has been elusive. Rapid intramolecular vibrational relaxation appears to redistribute energy throughout large molecules on timescales faster than dissociation so that any selectivity that may be injected by an excitation process is lost. The fragmentation of peptide ions activated by blackbody radiation, [1] IR multiphoton excitation, [2] UV laser excitation, [3][4][5] and collisions with gas-phase molecules or surfaces [6,7] involves vibrational excitation of precursor ions and consequently, production of similar types of daughter ions. The latter are primarily b-and y-type fragments as defined by the standard nomenclature shown in
The photodissociation by 157 nm light of singly- and doubly-charged peptide ions containing C- or N-terminal arginine residues was studied in a linear ion trap mass spectrometer. Singly-charged peptides yielded primarily x- and a-type ions, depending on the location of the arginine residue, along with some related side-chain fragments. These results are consistent with our previous work using a tandem time-of-flight (TOF) instrument with a vacuum matrix-assisted laser desorption/ionization (MALDI) source. Thus, the different internal energies of precursor ions in the two experiments seem to have little effect on their photofragmentation. For doubly-charged peptides, the dominant fragments observed in both photodissociation and collisionally induced dissociation (CID) experiments are b- and y-type ions. Preliminary experiments demonstrating fragmentation of multiply-charged ubiquitin ions by 157 nm photodissociation are also presented.
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