CRISPR-induced double-strand breaks trigger recombination between homologous chromosome arms

CRISPR–Cas9–based genome editing has transformed the life sciences, enabling virtually unlimited genetic manipulation of genomes: The RNA-guided Cas9 endonuclease cuts DNA at a specific target sequence and the resulting double-strand breaks are mended by one of the intrinsic cellular repair pathways. Imprecise double-strand repair will introduce random mutations such as indels or point mutations, whereas precise editing will restore or specifically edit the locus as mandated by an endogenous or exogenously provided template. Recent studies indicate that CRISPR-induced DNA cuts may also result in the exchange of genetic information between homologous chromosome arms. However, conclusive data of such recombination events in higher eukaryotes are lacking. Here, we show that in Drosophila, the detected Cas9-mediated editing events frequently resulted in germline-transmitted exchange of chromosome arms—often without indels. These findings demonstrate the feasibility of using the system for generating recombinants and also highlight an unforeseen risk of using CRISPR-Cas9 for therapeutic intervention

Multidifferential study of identified charged hadron distributions in ZZ-tagged jets in proton-proton collisions at s=\sqrt{s}=13 TeV

Jet fragmentation functions are measured for the first time in proton-proton collisions for charged pions, kaons, and protons within jets recoiling against a $Z$ boson. The charged-hadron distributions are studied longitudinally and transversely to the jet direction for jets with transverse momentum 20 $< p_{\textrm{T}} < 100$ GeV and in the pseudorapidity range $2.5 < \eta < 4$. The data sample was collected with the LHCb experiment at a center-of-mass energy of 13 TeV, corresponding to an integrated luminosity of 1.64 fb$^{-1}$. Triple differential distributions as a function of the hadron longitudinal momentum fraction, hadron transverse momentum, and jet transverse momentum are also measured for the first time. This helps constrain transverse-momentum-dependent fragmentation functions. Differences in the shapes and magnitudes of the measured distributions for the different hadron species provide insights into the hadronization process for jets predominantly initiated by light quarks.Comment: All figures and tables, along with machine-readable versions and any supplementary material and additional information, are available at https://cern.ch/lhcbproject/Publications/p/LHCb-PAPER-2022-013.html (LHCb public pages

Study of the B−→Λc+Λˉc−K−B^{-} \to \Lambda_{c}^{+} \bar{\Lambda}_{c}^{-} K^{-} decay

The decay $B^{-} \to \Lambda_{c}^{+} \bar{\Lambda}_{c}^{-} K^{-}$ is studied in proton-proton collisions at a center-of-mass energy of $\sqrt{s}=13$ TeV using data corresponding to an integrated luminosity of 5 $\mathrm{fb}^{-1}$ collected by the LHCb experiment. In the $\Lambda_{c}^+ K^{-}$ system, the $\Xi_{c}(2930)^{0}$ state observed at the BaBar and Belle experiments is resolved into two narrower states, $\Xi_{c}(2923)^{0}$ and $\Xi_{c}(2939)^{0}$, whose masses and widths are measured to be $$m(\Xi_{c}(2923)^{0}) = 2924.5 \pm 0.4 \pm 1.1 \,\mathrm{MeV}, \\ m(\Xi_{c}(2939)^{0}) = 2938.5 \pm 0.9 \pm 2.3 \,\mathrm{MeV}, \\ \Gamma(\Xi_{c}(2923)^{0}) = \phantom{000}4.8 \pm 0.9 \pm 1.5 \,\mathrm{MeV},\\ \Gamma(\Xi_{c}(2939)^{0}) = \phantom{00}11.0 \pm 1.9 \pm 7.5 \,\mathrm{MeV},$$ where the first uncertainties are statistical and the second systematic. The results are consistent with a previous LHCb measurement using a prompt $\Lambda_{c}^{+} K^{-}$ sample. Evidence of a new $\Xi_{c}(2880)^{0}$ state is found with a local significance of $3.8\,\sigma$, whose mass and width are measured to be $2881.8 \pm 3.1 \pm 8.5\,\mathrm{MeV}$ and $12.4 \pm 5.3 \pm 5.8 \,\mathrm{MeV}$, respectively. In addition, evidence of a new decay mode $\Xi_{c}(2790)^{0} \to \Lambda_{c}^{+} K^{-}$ is found with a significance of $3.7\,\sigma$. The relative branching fraction of $B^{-} \to \Lambda_{c}^{+} \bar{\Lambda}_{c}^{-} K^{-}$ with respect to the $B^{-} \to D^{+} D^{-} K^{-}$ decay is measured to be $2.36 \pm 0.11 \pm 0.22 \pm 0.25$, where the first uncertainty is statistical, the second systematic and the third originates from the branching fractions of charm hadron decays.Comment: All figures and tables, along with any supplementary material and additional information, are available at https://cern.ch/lhcbproject/Publications/p/LHCb-PAPER-2022-028.html (LHCb public pages

Measurement of the ratios of branching fractions R(D∗)\mathcal{R}(D^{*}) and R(D0)\mathcal{R}(D^{0})

The ratios of branching fractions $\mathcal{R}(D^{*})\equiv\mathcal{B}(\bar{B}\to D^{*}\tau^{-}\bar{\nu}_{\tau})/\mathcal{B}(\bar{B}\to D^{*}\mu^{-}\bar{\nu}_{\mu})$ and $\mathcal{R}(D^{0})\equiv\mathcal{B}(B^{-}\to D^{0}\tau^{-}\bar{\nu}_{\tau})/\mathcal{B}(B^{-}\to D^{0}\mu^{-}\bar{\nu}_{\mu})$ are measured, assuming isospin symmetry, using a sample of proton-proton collision data corresponding to 3.0 fb${ }^{-1}$ of integrated luminosity recorded by the LHCb experiment during 2011 and 2012. The tau lepton is identified in the decay mode $\tau^{-}\to\mu^{-}\nu_{\tau}\bar{\nu}_{\mu}$. The measured values are $\mathcal{R}(D^{*})=0.281\pm0.018\pm0.024$ and $\mathcal{R}(D^{0})=0.441\pm0.060\pm0.066$, where the first uncertainty is statistical and the second is systematic. The correlation between these measurements is $\rho=-0.43$. Results are consistent with the current average of these quantities and are at a combined 1.9 standard deviations from the predictions based on lepton flavor universality in the Standard Model.Comment: All figures and tables, along with any supplementary material and additional information, are available at https://cern.ch/lhcbproject/Publications/p/LHCb-PAPER-2022-039.html (LHCb public pages

Calibration of EGFP and mCherry wild type profiles.

<p>(A) Example of a 2D mask along the <i>wingless</i> expression domain in the posterior compartment of the pouch used to extract EGFP and mCherry pixel amplitude pairs. (B) Example of a 1D ROI used for profile extraction. The line was manually drawn parallel to the <i>wg</i> expression domain, around ten pixels into the dorsal compartment. (C) All the pixel pairs (absolute fluorescence mCherry versus EGFP) collected from the 2D mask (cf. A), resulting in a cone-shaped distribution. Data for all analyzed <i>allSEwt>EGFP>brk-tags</i> discs (n = 35) was pooled prior to analysis. (D) In black, the calibration profile obtained by a linear fit of the cleaned data (cf. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071224#pone.0071224.s003" target="_blank">Text S1</a> for more details). (E) Two representative <i>allSEwt>EGFP>brk-tags</i> (green) versus <i>allSEwt>mCherry-CAAX>brk-tags</i> (red) profiles (absolute values, expressed in arbitrary units) for the posterior half of the pouch prior to calibration. Overall, the mCherry profiles show higher levels than the EGFP profiles. The distance (x-axis) is always expressed in pixels (1 px = 0.664 µm). (F) Same profiles as in (E), after application of the calibration to the mCherry curves (the EGFP profiles remain unchanged). The profiles become quite similar in the medial region of interest. (G) Finally, we plot the difference δ = EGFP – mCherry. As expected, values are close to zero in the medial region (medial 50% of the posterior part of the pouch; marked by vertical line).</p

The incomplete derepression observed upon mutating all putative <i>SEs</i> still seems to be Dpp signaling dependent.

<p>(A) <i>EGFP</i> expression from the construct <i>allSEmut>EGFP>brk-tags</i> shows residual repression along the A/P compartment boundary independent of whether the construct is integrated at position 86Fb on chromosome III or at position 22A on chromosome II. (B) Ubiquitous <i>shn</i> expression from a genomic construct also cloned into the <i>pattB-P[acman]</i> integration vector (anti-HA staining) and ubiquitous <i>ubi-GFP-nls</i> expression, sequence was also cloned into the <i>pattB-P[acman]</i> integration vector. (C) <i>EGFP</i> expression patterns when expressed under the control of the endogenous <i>brk</i> regulatory region <i>(allSEwt>EGFP>brk-tags)</i> and upon mutating all potential <i>SEs (allSEmut>EGFP>brk-tags)</i>, the latter case resulting in a broadening of the Brk domain. The derepression does not take place throughout the disc. Following mutation of all the <i>SEs</i>, a slight overlap of <i>EGFP</i> expression and the anti-pMad staining can be observed. The overlap is not complete, indicating that in regions of high pMad (high Dpp signaling) there is still residual repression. (D) RNAi mediated <i>mad</i> knockdown in the dorsal compartment leads to uniform derepression of the <i>EGFP</i> readout in wing imaginal discs dissected from flies transgenic for the construct <i>allSEwt>EGFP>brk-tags.</i> The pMad staining is absent in the dorsal compartment where <i>mad</i> is knocked down via RNAi. (D’) Same as in (D), but for <i>allSEmut>EGFP>brk-tags.</i> (E) RNAi mediated <i>shn</i> knockdown in the dorsal compartment also leads to uniform derepression of the <i>EGFP</i> readout, again in wing imaginal discs dissected from flies transgenic for the construct <i>allSEwt>EGFP>brk-tags</i>. The anti-GAL4 staining marks the RNAi expression domain. (E’) Same as in (E), but for <i>allSEmut>EGFP>brk-tags</i>. Scale bars: 50 µm. <i>UAS-shn RNAi</i> and <i>UAS-mad RNAi</i>: pictures taken with identical magnification.</p

Quantification of the individual EGFP profiles for the different constructs, relative to the internal mCherry wild type control.

<p>(A) Four examples of individual wing discs carrying the indicated constructs. As in Fig. 3G, the black lines represent the difference between the EGFP and the mCherry profiles (δ = EGFP – mCherry). The vertical line again marks the medial 50% of the posterior part of the pouch. (B) Summary of the results for all the different constructs. We show, for each construct, the absolute area (medial 50% of the posterior part of the pouch) below the black δ curve (cf. A) divided by the absolute area below the red mCherry curve. Error bars represent ± two times the standard deviation for the corresponding construct. For each construct, between 6 <i>(SE10wt)</i> and 35 <i>(allSEwt)</i> individual wing discs were analyzed (<i>allSEwt</i> n = 35, <i>SE3-8wt</i> n = 14, <i>SE3&4&5&7&8wt</i> n = 12, <i>SE3-6wt</i> n = 9, <i>SE3&4&8wt</i> n = 11, <i>SE3&4wt</i> n = 7, <i>SE4wt</i> n = 8, <i>SE10wt</i> n = 6, <i>SE1&2wt</i> n = 13, <i>allSEmut</i> n = 9). Taking into account only 50% of the profile gives the best results (lowest standard deviations). Different cases (namely 70% or 100%) are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071224#pone.0071224.s003" target="_blank">Text S1</a>. The color code employed in this Figure is reused in the additional plots that can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071224#pone.0071224.s003" target="_blank">Text S1</a>.</p

Mutating <i>SEs</i> that show an even more degenerate consensus increases <i>brk</i> derepression.

<p>(A) Schematic overview of the <i>brk</i> locus. The four fragments covering the 13 <i>SEs</i>, as well as the additionally identified <i>SEs</i>, which show a single bp substitution if compared to the perfect consensus are indicated (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071224#pone.0071224-Gao1" target="_blank">[2]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071224#pone.0071224-Pyrowolakis1" target="_blank">[8]</a>; <i>SEm3</i>, <i>SEm1</i> and <i>SEm3-2)</i>. (B)-(D) Genomic fragments covering the wild type <i>SE</i> combinations <i>SE1&2</i>, <i>SE3-8</i>, <i>SE9-12</i>. (E)-(G) Similar fragments as shown in (B)-(D) after mutating the <i>SEs</i>. (H) The genomic fragment featuring mutated <i>SE1&2</i> as well as additional mutations in <i>SEm3</i> and <i>SEm1</i>. The fragment featuring <i>SE13</i> is not shown, as no expression could be observed with <i>SE13mut</i>. Anti-β-Gal stainings were performed. Scale bar: 50 µm. Pictures were taken with constant confocal settings.</p

Mutating the <i>SEs</i> results in a medial expansion of the <i>EGFP</i> expression domain.

<p><i>EGFP</i> expression patterns resulting from different subsets of functional and mutated <i>SEs</i>. The expression pattern seems to be a function of both the number and identity of the functional <i>SEs</i>. Genotypes are indicated. The number of functional <i>SEs</i> present in the constructs is given in red. (A)-(J) Expression patterns in the wing imaginal disc. (K)-(L) Expression patterns in the eye disc. Scale bar 50 µm. Pictures taken with constant confocal settings.</p