Objective

To systematically investigate the effect of CT localizer radiograph acquisition on the tube current modulation and thus radiation dose of the subsequent diagnostic scan.

Methods

Localizer radiographs of an abdominal section CT phantom were taken, and the resulting volume CT dose index (CTDIvol) for the diagnostic scan was recorded. Variables included tube potential, the phantom's alignment within the CT scanner gantry in both the vertical and horizontal directions and the X-ray source angle at which the localizer was acquired.

Results

Diagnostic scan CTDIvol decreased with increasing tube potential. Vertical (table height) movement was found to affect radiation dose more than horizontal movement, with ±50 mm table movement resulting in a standard deviation in the diagnostic scan CTDIvol of 4.4 mGy, compared with 2.5 mGy with ±50 mm horizontal movement. Correspondingly, localizer angles of 90° or 270° (3 o'clock and 9 o'clock X-ray source positions) were less sensitive overall to alignment errors, with a standard deviation of 2.5 mGy, compared with a 0° or 180° angle, which had a standard deviation of 3.8 mGy.

Conclusion

To achieve a consistently optimized radiation dose, the localizer protocol should be paired with the diagnostic acquisition protocol. A final acquisition angle of 90° should be used when possible to minimize dose variation resulting from alignment errors.

Advances in knowledge

Localizer parameters that affect radiation output were identified for this scanner system. The importance of tube potential and acquisition angle was highlighted.

CRISPR-Cas9 is the state-of-the-art technology for editing and manipulating nucleic acids. However, the occurrence of off-target mutations can limit its applicability. Here, all-atom enhanced molecular dynamics (MD) simulations-using Gaussian accelerated MD (GaMD)-are used to decipher the mechanism of off-target binding at the molecular level. GaMD reveals that base pair mismatches in the target DNA at distal sites with respect to the protospacer adjacent motif (PAM) can induce an extended opening of the RNA:DNA heteroduplex, which leads to newly formed interactions between the unwound DNA and the L2 loop of the catalytic HNH domain. These conserved interactions constitute a "lock" effectively decreasing the conformational freedom of the HNH domain and hampering its activation for cleavage. Remarkably, depending on their positions at PAM distal sites, DNA mismatches responsible for off-target cleavages are unable to "lock" the HNH domain, thereby leading to the unselective cleavage of DNA sequences. In consistency with the available experimental data, the ability to "lock" the catalytic HNH domain in an inactive "conformational checkpoint" is shown to be a key determinant in the onset of off-target effects. This mechanistic rationale contributes in clarifying a long lasting open issue in the CRISPR-Cas9 function and poses the foundation for designing novel and more specific Cas9 variants, which could be obtained by magnifying the "locking" interactions between HNH and the target DNA in the presence of any incorrect off-target sequence, thus preventing undesired cleavages.

CRISPR-Cas systems provide microbes with adaptive immunity to infectious nucleic acids and are widely employed as genome editing tools. These tools use RNA-guided Cas proteins whose large size (950 to 1400 amino acids) has been considered essential to their specific DNA- or RNA-targeting activities. Here we present a set of CRISPR-Cas systems from uncultivated archaea that contain Cas14, a family of exceptionally compact RNA-guided nucleases (400 to 700 amino acids). Despite their small size, Cas14 proteins are capable of targeted single-stranded DNA (ssDNA) cleavage without restrictive sequence requirements. Moreover, target recognition by Cas14 triggers nonspecific cutting of ssDNA molecules, an activity that enables high-fidelity single-nucleotide polymorphism genotyping (Cas14-DETECTR). Metagenomic data show that multiple CRISPR-Cas14 systems evolved independently and suggest a potential evolutionary origin of single-effector CRISPR-based adaptive immunity.