Engineered CRISPR-Cas12a for higher-order combinatorial chromatin perturbations.

Multiplexed genetic perturbations are critical for testing functional interactions among coding or non-coding genetic elements. Compared to double-stranded DNA cutting, repressive chromatin formation using CRISPR interference (CRISPRi) avoids genotoxicity and is more effective for perturbing non-coding regulatory elements in pooled assays. However, current CRISPRi pooled screening approaches are limited to targeting one to three genomic sites per cell. We engineer an Acidaminococcus Cas12a (AsCas12a) variant, multiplexed transcriptional interference AsCas12a (multiAsCas12a), that incorporates R1226A, a mutation that stabilizes the ribonucleoprotein-DNA complex via DNA nicking. The multiAsCas12a-KRAB fusion improves CRISPRi activity over DNase-dead AsCas12a-KRAB fusions, often rescuing the activities of lentivirally delivered CRISPR RNAs (crRNA) that are inactive when used with the latter. multiAsCas12a-KRAB supports CRISPRi using 6-plex crRNA arrays in high-throughput pooled screens. Using multiAsCas12a-KRAB, we discover enhancer elements and dissect the combinatorial function of cis-regulatory elements in human cells. These results instantiate a group testing framework for efficiently surveying numerous combinations of chromatin perturbations for biological discovery and engineering.

Higher-order combinatorial chromatin perturbations by engineered CRISPR-Cas12a for functional genomics.

Multiplexed genetic perturbations are critical for testing functional interactions among coding or non-coding genetic elements. Compared to double-stranded DNA cutting, repressive chromatin formation using CRISPR interference (CRISPRi) avoids genotoxicity and is more effective for perturbing non-coding regulatory elements in pooled assays. However, current CRISPRi pooled screening approaches are limited to targeting 1-3 genomic sites per cell. To develop a tool for higher-order ( > 3) combinatorial targeting of genomic sites with CRISPRi in functional genomics screens, we engineered an Acidaminococcus Cas12a variant -- referred to as mul tiplexed transcriptional interference AsCas12a (multiAsCas12a). multiAsCas12a incorporates a key mutation, R1226A, motivated by the hypothesis of nicking-induced stabilization of the ribonucleoprotein:DNA complex for improving CRISPRi activity. multiAsCas12a significantly outperforms prior state-of-the-art Cas12a variants in combinatorial CRISPRi targeting using high-order multiplexed arrays of lentivirally transduced CRISPR RNAs (crRNA), including in high-throughput pooled screens using 6-plex crRNA array libraries. Using multiAsCas12a CRISPRi, we discover new enhancer elements and dissect the combinatorial function of cis-regulatory elements. These results instantiate a group testing framework for efficiently surveying potentially numerous combinations of chromatin perturbations for biological discovery and engineering.

Acute proximal occlusion of a nonaneurysmal abdominal aorta and renal arteries detected by renal imaging.

This report describes an unusual case of extensive vascular thrombosis involving the abdominal aorta and its branches. An 81-year-old man was admitted for anuric acute renal failure and congestive heart failure. An initial renal scan, performed to assess for the possibility of renal arterial embolus, showed scintigraphic evidence of obstruction of the proximal abdominal aorta, as well as markedly decreased perfusion to both kidneys and to the liver and spleen. The patient's condition progressively deteriorated and he expired. An autopsy showed total thrombotic occlusion of a mildly atherosclerotic nonaneurysmal abdominal aorta extending from the level of the superior mesenteric artery distally to the iliac arteries. There was involvement of the renal arteries and the splenic and superior mesenteric arteries by thrombosis. Thus, renal scintigraphy accurately detected the level of obstruction, which was further confirmed by autopsy.

Anorectal bleeding vs penile activity. A potential diagnostic problem.

Intrapenile blood pool activity may be a source of artefact in interpreting a gastrointestinal bleeding study employing Tc-99m sulfur colloid or erythrocytes. Proper positioning should avoid a false reading of rectal bleeding.

Erythropoietic protoporphyria: juvenile protoporphyrin hepatopathy cirrhosis and death.

Severe hepatic cirrhosis and failure in erythropoietic protoporphyria is rare. An 11-year-old boy is described who developed protoporphyrin hepatopathy (protoporphyrin 5.75 mg/gm liver wet weight), cirrhosis, and liver failure and died.

New neurological findings in trisomy 13.

The patient in this report had many of the classic neuropathologic stigmata of trisomy 13, including retinal dysplasia, arrhinencephaly, holoprosencephaly, single external nare and granular cell heterotopias in the cerebellum and microthalmia. In addition, several new findings apparently were present in this case. The neuropathologic entities were as follows: (1) herniation of the cochlear nuclei into the eighth cranial nerve bilaterally to the transition of central to peripheral myelin; (2) gray matter present in eleventh cranial nerve bilaterally; (3) arteriovenous malformations of letpomeningeal and intracerebral vessels; (4) arachnoid cyst at the cauda equina; and (5) retinal pigment epithelium within the optic nerve.