28Pooled CRISPR-Cas9 screens have recently emerged as a powerful method for functionally 29 characterizing regulatory elements in the non-coding genome, but off-target effects in these 30 experiments have not been systematically evaluated. Here, we conducted a genome-scale screen 31 for essential CTCF loop anchors in the K562 leukemia cell line. Surprisingly, the primary drivers 32 of signal in this screen were single guide RNAs (sgRNAs) with low specificity scores. After 33 removing these guides, we found that there were no CTCF loop anchors critical for cell growth. 34We also observed this effect in an independent screen fine-mapping the core motifs in enhancers 35 of the GATA1 gene. We then conducted screens in parallel with CRISPRi and CRISPRa, which 36 do not induce DNA damage, and found that an unexpected and distinct set of off-targets also 37 caused strong confounding growth effects with these epigenome-editing platforms. Promisingly, 38 strict filtering of CRISPRi libraries using GuideScan specificity scores removed these confounded 39 sgRNAs and allowed for the identification of essential enhancers, which we validated extensively. 40Together, our results show off-target activity can severely limit identification of essential functional 41 motifs by active Cas9, while strictly filtered CRISPRi screens can be reliably used for assaying 42 larger regulatory elements. 43 4 indels would be expected to have the same effect on the gene (i.e. a complete knockout), making 69 the selection of highly specific sgRNAs relatively straightforward 34,[36][37][38] . On the other hand, 70 screens of non-coding elements that use active Cas9 often require the use of lower specificity 71 sgRNAs because regulatory elements, such as individual TF motifs, present a more narrow 72 targeting window from which fewer sgRNAs may be selected. 73 74 Despite these challenges, CRISPR-Cas9 screens present an opportunity to systematically 75 perturb and functionally characterize non-coding elements that could not be studied with earlier 76 high-throughput technologies like shRNAs, gene traps, or ORF libraries. One class of candidate 77 cis-regulatory elements (ccREs) that have not been functionally dissected in a high-throughput 78 manner are CTCF binding sites in chromatin loop anchors. CTCF binding sites are enriched at 79 the boundaries that partition interphase vertebrate genomes into TADs (Topologically Associated 80 Domains) 39,40 , and pairs of convergently oriented CTCF motifs are enriched at the anchors of 81 chromatin loops 40-42 . These chromatin loops and TADs are thought to constrain enhancer-82 promoter interactions, adding a layer of specificity to the cis-regulatory wiring that connects genes 83 with distal regulatory elements. CRISPR-mediated deletions and inversions of individual CTCF 84 sites have been shown to result in reorganization of TADs 41 and occasionally in changes in gene 85 expression [43][44][45] . Moreover, disruptions of CTCF occupancy have been suggested to be involved 86 in tumorigenesis by leading to ...

Detecting and mitigating off-target activity is critical to the practical application of CRISPR-mediated genome and epigenome editing. While numerous methods have been developed to map Cas9 binding specificity genome-wide, they are generally time-consuming and/or expensive, and not applicable to catalytically dead CRISPR enzymes. We have developed a rapid, inexpensive, and facile assay for identifying off-target CRISPR enzyme binding and cleavage by chemically mapping the unwound single-stranded DNA structure formed upon binding of a sgRNA-loaded Cas9 protein (''CasKAS''). We demonstrate this method in both in vitro and in vivo contexts.

Background: The COVID-19 pandemic followed a unique trajectory in Eastern Europe compared to other heavily affected regions, with most countries there only experiencing a major surge of cases and deaths towards the end of 2020 after a relatively uneventful first half of the year. However, the consequences of that surge have not received as much attention as the situation in Western countries. Bulgaria, even though it has been one of the most heavily affected countries, has been one of those neglected cases. Methods: We use mortality and mobility data from Eurostat, official governmental and other sources to examine the development and impact of the COVID-19 pandemic in Bulgaria and other European countries. Results: We find a very high level of excess mortality in Eastern European countries measured by several metrics including excess mortality rate (EMR), P-scores and potential years of life lost. By the last metric Eastern Europe emerges as the hardest hit region by the pandemic in Europe in 2020. With a record EMR at ~0.25% and a strikingly large and mostly unique to it mortality rate in the working age demographics, Bulgaria emerges as one of the most affected countries in Eastern Europe. The high excess mortality in Bulgaria correlates with insufficient intensity of testing and with delayed imposition of lockdown measures. We also find major geographic and demographic disparities within the country, with considerably lower mortality observed in major cities relative to more remote areas (likely due to disparities in the availability of medical resources). Analysis of the course of the epidemic revealed that individual mobility measures were predictive of the eventual decline in cases and deaths. However, while mobility declined as a result of the imposition of a lockdown, it already trended downwards before such measures were introduced, which resulted in a reduction of deaths independent of the effect of restrictions. Conclusions: Large excess mortality and high numbers of potential years of life lost are observed as a result of the COVID pandemic in Bulgaria, as well as in several other countries in Eastern Europe. Significant delays in the imposition of stringent mobility-reducing measures combined with a lack of medical resources likely caused a substantial loss of life, including in the working age population.