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Creators/Authors contains: "Zou, Roger S."

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  1. Abstract

    Direct visualization of point mutations in situ can be informative for studying genetic diseases and nuclear biology. We describe a direct hybridization genome imaging method with single-nucleotide sensitivity, single guide genome oligopaint via local denaturation fluorescence in situ hybridization (sgGOLDFISH), which leverages the high cleavage specificity of eSpCas9(1.1) variant combined with a rationally designed guide RNA to load a superhelicase and reveal probe binding sites through local denaturation. The guide RNA carries an intentionally introduced mismatch so that while wild-type target DNA sequence can be efficiently cleaved, a mutant sequence with an additional mismatch (e.g., caused by a point mutation) cannot be cleaved. Because sgGOLDFISH relies on genomic DNA being cleaved by Cas9 to reveal probe binding sites, the probes will only label the wild-type sequence but not the mutant sequence. Therefore, sgGOLDFISH has the sensitivity to differentiate the wild-type and mutant sequences differing by only a single base pair. Using sgGOLDFISH, we identify base-editor-modified and unmodified progeroid fibroblasts from a heterogeneous population, validate the identification through progerin immunofluorescence, and demonstrate accurate sub-nuclear localization of point mutations.

  2. Abstract

    Polymerase Chain Reaction (PCR) is an essential method in molecular diagnostics and life sciences. PCR requires thermal cycling for heating the DNA for strand separation and cooling it for replication. The process uses a specialized hardware and exposes biomolecules to temperatures above 95 °C. Here, we engineer a PcrA M6 helicase with enhanced speed and processivity to replace the heating step by enzymatic DNA unwinding while retaining desired PCR characteristics. We name this isothermal amplification method SHARP (SSB-Helicase Assisted Rapid PCR) because it uses the engineered helicase and single-stranded DNA binding protein (SSB) in addition to standard PCR reagents. SHARP can generate amplicons with lengths of up to 6000 base pairs. SHARP can produce functional DNA, a plasmid that imparts cells with antibiotic resistance, and can amplify specific fragments from genomic DNA of human cells. We further use SHARP to assess the outcome of CRISPR-Cas9 editing at endogenous genomic sites.

  3. Abstract

    Here we present an approach that combines a clustered regularly interspaced short palindromic repeats (CRISPR) system that simultaneously targets hundreds of epigenetically diverse endogenous genomic sites with high-throughput sequencing to measure Cas9 dynamics and cellular responses at scale. This massive multiplexing of CRISPR is enabled by means of multi-target guide RNAs (mgRNAs), degenerate guide RNAs that direct Cas9 to a pre-determined number of well-mapped sites. mgRNAs uncovered generalizable insights into Cas9 binding and cleavage, revealing rapid post-cleavage Cas9 departure and repair factor loading at protospacer adjacent motif-proximal genomic DNA. Moreover, by bypassing confounding effects from guide RNA sequence, mgRNAs unveiled that Cas9 binding is enhanced at chromatin-accessible regions, and cleavage by bound Cas9 is more efficient near transcribed regions. Combined with light-mediated activation and deactivation of Cas9 activity, mgRNAs further enabled high-throughput study of the cellular response to double-strand breaks with high temporal resolution, revealing the presence, extent (under 2 kb) and kinetics (~1 h) of reversible DNA damage-induced chromatin decompaction. Altogether, this work establishes mgRNAs as a generalizable platform for multiplexing CRISPR and advances our understanding of intracellular Cas9 activity and the DNA damage response at endogenous loci.

  4. Abstract

    Nucleic acid detection is essential for numerous biomedical applications, but often requires complex protocols and/or suffers false-positive readouts. Here, we describe SENTINEL, an approach that combines isothermal amplification with a sequence-specific degradation method to detect nucleic acids with high sensitivity and sequence-specificity. Target single-stranded RNA or double-stranded DNA molecules are amplified by loop-mediated isothermal amplification (LAMP) and subsequently degraded by the combined action of lambda exonuclease and a sequence-specific DNA endonuclease (e.g., Cas9). By combining the sensitivity of LAMP with the precision of DNA endonucleases, the protocol achieves attomolar limits of detection while differentiating between sequences that differ by only one or two base pairs. The protocol requires less than an hour to complete using a 65 °C heat block and fluorometer, and detects SARS-CoV-2 virus particles in human saliva and nasopharyngeal swabs with high sensitivity.

  5. CRISPR-Cas systems provide versatile tools for programmable genome editing. Here, we developed a caged RNA strategy that allows Cas9 to bind DNA but not cleave until light-induced activation. This approach, referred to as very fast CRISPR (vfCRISPR), creates double-strand breaks (DSBs) at the submicrometer and second scales. Synchronized cleavage improved kinetic analysis of DNA repair, revealing that cells respond to Cas9-induced DSBs within minutes and can retain MRE11 after DNA ligation. Phosphorylation of H2AX after DNA damage propagated more than 100 kilobases per minute, reaching up to 30 megabases. Using single-cell fluorescence imaging, we characterized multiple cycles of 53BP1 repair foci formation and dissolution, with the first cycle taking longer than subsequent cycles and its duration modulated by inhibition of repair. Imaging-guided subcellular Cas9 activation further facilitated genomic manipulation with single-allele resolution. vfCRISPR enables DNA-repair studies at high resolution in space, time, and genomic coordinates.