Combining CRISPR-Cas9 and Proximity Labeling to Illuminate Chromatin Composition, Organization, and Regulation

A bacterial and archaeal adaptive immune system, clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas), has recently been engineered for genome editing. This RNA-guided platform has simplified genetic manipulation and holds promise for therapeutic applications. However, off-target editing has been one of the major concerns of the commonly used Streptococcus pyogenes Cas9 (SpyCas9). Despite extensive enzyme engineering to reduce off-target editing of SpyCas9, we have turned to nature and uncovered a Cas9 ortholog from Neisseria meningitidis (Nme) with high fidelity. In the first part of my thesis, we have systematically characterized Nme1Cas9 for engineering mammalian genomes and demonstrated its high specificity by genome-wide off-targeting detection methods in vitro and in cellulo, and thus provided a new platform for accurate genome editing. Due to its flexibility, CRISPR is becoming a versatile tool not only for genome editing, but also for chromatin manipulation. These alternative applications are possible because of the programmable targeting capacity of catalytically dead Cas9 (dCas9). In the second part of my thesis, we have combined dCas9 with the engineered plant enzyme ascorbate peroxidase (APEX2) to develop a proteomic method called dCas9-APEX2 biotinylation at genomic elements by restricted spatial tagging (C-BERST). Relying on the spatially restricted, fast biotin labeling of proteins near defined genomic loci, C-BERST enables the high-throughput identification of known telomere- and centromere- associated proteomes and novel factors. Furthermore, we have extended C-BERST to map the c-fos promoter and gained new insights regarding the dynamic transcriptional regulation process. Taken together, C-BERST can advance our understanding of chromatin regulators and their roles in nuclear and chromosome biology

Leading-twist parton distribution amplitudes of S-wave heavy-quarkonia

The leading-twist parton distribution amplitudes (PDAs) of ground-state $^1S_0$ and $^3S_1$ $c\bar c$- and $b\bar b$-quarkonia are calculated using a symmetry-preserving continuum treatment of the meson bound-state problem which unifies the properties of these heavy-quark systems with those of light-quark bound-states, including QCD's Goldstone modes. Analysing the evolution of $^1S_0$ and $^3S_1$ PDAs with current-quark mass, $\hat m_q$, increasing away from the chiral limit, it is found that in all cases there is a value of $\hat m_q$ for which the PDA matches the asymptotic form appropriate to QCD's conformal limit and hence is insensitive to changes in renormalisation scale, $\zeta$. This mass lies just above that associated with the $s$-quark. At current-quark masses associated with heavy-quarkonia, on the other hand, the PDAs are piecewise convex-concave-convex. They are much narrower than the asymptotic distribution on a large domain of $\zeta$; but nonetheless deviate noticeably from $\varphi_{Q\bar Q}(x) = \delta(x-1/2)$, which is the result in the static-quark limit. There are also material differences between $^1S_0$ and $^3S_1$ PDAs, and between the PDAs for different vector-meson polarisations, which vanish slowly with increasing $\zeta$. An analysis of moments of the root-mean-square relative-velocity, $\langle v^{2m}\rangle$, in $^1S_0$ and $^3S_1$ systems reveals that $\langle v^4\rangle$-contributions may be needed in order to obtain a reliable estimate of matrix elements using such an expansion, especially for processes involving heavy pseudoscalar quarkonia.Comment: 6 pages, 2 figures, 3 table

Exposing strangeness: projections for kaon electromagnetic form factors

A continuum approach to the kaon and pion bound-state problems is used to reveal their electromagnetic structure. For both systems, when used with parton distribution amplitudes appropriate to the scale of the experiment, Standard Model hard-scattering formulae are accurate to within 25% at momentum transfers $Q^2 \approx 8\,$GeV$^2$. There are measurable differences between the distribution of strange and normal matter within the kaons, e.g. the ratio of their separate contributions reaches a peak value of $1.5$ at $Q^2 \approx 6\,$GeV$^2$. Its subsequent $Q^2$-evolution is accurately described by the hard scattering formulae. Projections for kaon and pion form factors at timelike momenta beyond the resonance region are also presented. These results and projections should prove useful in planning next-generation experiments.Comment: 7 pages, 4 figure

Parton distribution amplitudes of light vector mesons

A rainbow-ladder truncation of QCD's Dyson-Schwinger equations is used to calculate rho- and phi-meson valence-quark (twist-two parton) distribution amplitudes (PDAs) via a light-front projection of their Bethe-Salpeter wave functions, which possess S- and D-wave components of comparable size in the meson rest frame. All computed PDAs are broad concave functions, whose dilation with respect to the asymptotic distribution is an expression of dynamical chiral symmetry breaking. The PDAs can be used to define an ordering of valence-quark light-front spatial-extent within mesons: this size is smallest within the pion and increases through the perp-polarisation to the parallel-polarisation of the vector mesons; effects associated with the breaking of SU(3)-flavour symmetry are significantly smaller than those associated with altering the polarisation of vector mesons. Notably, the predicted pointwise behaviour of the rho-meson PDAs is in quantitative agreement with that inferred recently via an analysis of diffractive vector-meson photoproduction experiments.Comment: 13 pages, 4 figures, 4 table

Zero mode in a strongly coupled quark gluon plasma

In connection with massless two-flavour QCD, we analyse the chiral symmetry restoring phase transition using three distinct gluon-quark vertices and two different assumptions about the long-range part of the quark-quark interaction. In each case, we solve the gap equation, locate the transition temperature T_c, and use the maximum entropy method to extract the dressed-quark spectral function at T>T_c. Our best estimate for the chiral transition temperature is T_c=(147 +/- 8)MeV; and the deconfinement transition is coincident. For temperatures markedly above T_c, we find a spectral density that is consistent with those produced using a hard thermal loop expansion, exhibiting both a normal and plasmino mode. On a domain T\in[T_c,T_s], with T_s approximately 1.5T_c, however, with each of the six kernels we considered, the spectral function contains a significant additional feature. Namely, it displays a third peak, associated with a zero mode, which is essentially nonperturbative in origin and dominates the spectral function at T=T_c. We suggest that the existence of this mode is a signal for the formation of a strongly-coupled quark-gluon plasma and that this strongly-interacting state of matter is likely a distinctive feature of the QCD phase transition.Comment: 11 pages, 5 figures, 1 tabl

Temperature-dependent contact of weakly interacting single-component Fermi gases and loss rate of degenerate polar molecules

Motivated by the experimental realization of single-component degenerate Fermi gases of polar ground state KRb molecules with intrinsic two-body losses [L. De Marco, G. Valtolina, K. Matsuda, W. G. Tobias, J. P. Covey, and J. Ye, A degenerate Fermi gas of polar molecules, Science 363, 853 (2019)], this work studies the finite-temperature loss rate of single-component Fermi gases with weak interactions. First, we establish a relationship between the two-body loss rate and the $p$-wave contact. Second, we evaluate the contact of the homogeneous system in the low-temperature regime using $p$-wave Fermi liquid theory and in the high-temperature regime using the second-order virial expansion. Third, conjecturing that there are no phase transitions between the two temperature regimes, we smoothly interpolate the results to intermediate temperatures. It is found that the contact is constant at temperatures close to zero and increases first quadratically with increasing temperature and finally -- in agreement with the Bethe-Wigner threshold law -- linearly at high temperatures. Fourth, applying the local-density approximation, we obtain the loss-rate coefficient for the harmonically trapped system, reproducing the experimental KRb loss measurements within a unified theoretical framework over a wide temperature regime without fitting parameters. Our results for the contact are not only applicable to molecular $p$-wave gases but also to atomic single-component Fermi gases, such as 40K and 6Li

Phase diagram and thermal properties of strong-interaction matter

We introduce a novel procedure for computing the (mu,T)-dependent pressure in continuum QCD; and therefrom obtain a complex phase diagram and predictions for thermal properties of the system, providing the in-medium behaviour of the trace anomaly, speed of sound, latent heat and heat capacity.Comment: 6 pages, 4 figures. Minor amendments in the version accepted for publicatio

Casimir effect for the massless Dirac field in two-dimensional Reissner-Nordstr\"{o}m spacetime

In this paper, the two-dimensional Reissner-Nordstr\"{o}m black hole is considered as a system of the Casimir type. In this background the Casimir effect for the massless Dirac field is discussed. The massless Dirac field is confined between two ``parallel plates'' separated by a distance $L$ and there is no particle current drilling through the boundaries. The vacuum expectation values of the stress tensor of the massless Dirac field at infinity are calculated separately in the Boulware state, the Hartle-Hawking state and the Unruh state.Comment: 10 pages, no figure. Accepted for publication in IJMP