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Programmable double-strand DNA breaks (DSBs) can be harnessed for precision genome editing through manipulation of the homology-directed repair (HDR) pathway. However, end-joining repair pathways often outcompete HDR and introduce insertions and deletions of bases (indels) at the DSB site, decreasing precision outcomes. It has been shown that indel sequences for a given DSB site are reproducible and can even be predicted. Here, we report a general strategy (the “double tap” method) to improve HDR-mediated precision genome editing efficiencies that takes advantage of the reproducible nature of indel sequences. The method simply involves the use of multiple gRNAs: a primary gRNA that targets the wild-type genomic sequence, and one or more secondary gRNAs that target the most common indel sequence(s), which in effect provides a “second chance” at HDR-mediated editing. This proof-of-principle study presents the double tap method as a simple yet effective option for enhancing precision editing in mammalian cells.

 

Homing CRISPR gene drives could aid in curbing the spread of vector-borne diseases and controlling crop pest and invasive species populations due to an inheritance rate that surpasses Mendelian laws. However, this technology suffers from resistance alleles formed when the drive-induced DNA break is repaired by error-prone pathways, which creates mutations that disrupt the gRNA recognition sequence and prevent further gene-drive propagation. Here, we attempt to counteract this by encoding additional gRNAs that target the most commonly generated resistance alleles into the gene drive, allowing a second opportunity at gene-drive conversion. Our presented “double-tap” strategy improved drive efficiency by recycling resistance alleles. The double-tap drive also efficiently spreads in caged populations, outperforming the control drive. Overall, this double-tap strategy can be readily implemented in any CRISPR-based gene drive to improve performance, and similar approaches could benefit other systems suffering from low HDR frequencies, such as mammalian cells or mouse germline transformations.

 

The novel 9.7m Schwarzschild-Couder Telescope (SCT), utilizing aspheric dual-mirror optical system, has been constructed as a prototype medium size x-ray telescope for the Cherenkov Telescope Array (CTA) observatory. The prototype SCT (pSCT) is designed to achieve simultaneously the wide (≥ 8°) field of view and the superior imaging resolution (0.067 per pixel) to significantly improve scientific capabilities of the observatory in conducting the sky surveys, the follow-up observations of multi-messenger transients with poorly known initial localization and the morphology studies of x-ray sources with angular extent. In this submission, we describe the hardware and software implementations of the telescope optical system as well as the methods specifically developed to align its complex optical system, in which both primary and secondary mirrors are segmented. The pSCT has detected Crab Nebula in June 2020 during ongoing commissioning, which was delayed due to worldwide pandemic and is not yet completed. Verification of pSCT performance is continuing and further improvement of optical alignment is anticipated. 

For the first time in the history of ground-based x-ray astronomy, the on-axis performance of the dual mirror, aspheric, aplanatic Schwarzschild-Couder optical system has been demonstrated in a 9:7-m aperture imaging atmospheric Cherenkov telescope. The novel design of the prototype Schwarzschild-Couder Telescope (pSCT) is motivated by the need of the next-generation Cherenkov Telescope Array (CTA) observatory to have the ability to perform wide (>=8°) field-of-view observations simultaneously with superior imaging of atmospheric cascades (resolution of 0:067 per pixel or better). The pSCT design, if implemented in the CTA installation, has the potential to improve significantly both the x-ray angular resolution and the off-axis sensitivity of the observatory, reaching nearly the theoretical limit of the technique and thereby making a major impact on the CTA observatory sky survey programs, follow-up observations of multi-messenger transients with poorly known initial localization, as well as on the spatially resolved spectroscopic studies of extended x-ray sources. This contribution reports on the initial alignment procedures and point-spread-function results for the challenging segmented aspheric primary and secondary mirrors of the pSCT.