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.

Crystallization and preliminary X-ray diffraction analysis of MspI restriction endonuclease in complex with its cognate DNA.

The MspI restriction endonuclease is a type II restriction enzyme. Unlike all other restriction enzymes with known structures, MspI recognizes the palindromic tetranucleotide sequence 5'-C/CGG and cleaves it as indicated by the '/' to produce DNA products with 5' two-base overhangs. Owing to the nature of its cleavage pattern, it is likely that MspI would represent a new structural class of restriction endonucleases. Crystals of the dimeric MspI restriction enzyme bound to a duplex DNA molecule containing the specific recognition sequence have been obtained by vapor-diffusion techniques in the presence of polyethylene glycol as precipitant. The crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 50.2, b = 131.6, c = 59.3 A, beta = 109.7 degrees. The crystals contain one dimeric complex in the asymmetric unit. A complete native data set has been collected to a resolution of 2.05 A by cryo-crystallographic methods, with an R(merge) of 4.0%.

Identification of a CD4 domain required for interleukin-16 binding and lymphocyte activation.

Interleukin-16 (IL-16) activates CD4(+) cells, possibly by direct interaction with CD4. IL-16 structure and function are highly conserved across species, suggesting similar conservation of a putative IL-16 binding site on CD4. Comparison of the human CD4 amino acid sequence with that of several different species revealed that immunoglobulin-like domain 4 is the most conserved extracellular region. Potential interaction of this domain with IL-16 was studied by testing murine D4 sequence-based oligopeptides for inhibition of IL-16 chemoattractant activity and inhibition of IL-16 binding to CD4 in vitro. Three contiguous 12-residue D4 region peptides (designated A, B, and C) blocked IL-16 chemoattractant activity, with peptide B the most potent. Peptides A and B were synergistic for inhibition, but peptide C was not. Peptides A and B also blocked IL-16 binding to CD4 in vitro, whereas peptide C did not. CD4, in addition to its known function as a receptor for major histocompatibility complex class II, contains a binding site for IL-16 in the D4 domain. The D4 residues required for IL-16 binding overlap those previously shown to participate in CD4-CD4 dimerization following class II major histocompatibility complex binding, providing a mechanistic explanation for the known function of IL-16 to inhibit the mixed lymphocyte reaction.

Cervical intraepithelial neoplasia and 'wary' atypia: a study of colposcopic, histological and cytological characteristics.

Colposcopic, histological and cytological characteristics of 168 consecutive patients attending a colposcopy clinic were reviewed. A group of patients demonstrated a cluster of histological features previously related to possible human papilloma virus infection of cervical epithelium, including particularly koilocytosis, double nucleation and dyskeratosis. These patients had a significantly greater occurrence of colposcopic 'finger-like' lesion outline and colposcopic impression of warty change, but not of other specific colposcopic features. Over two-thirds of patients with histological warty features did not show these colposcopic changes. Cytological changes previously related to warty lesions did not occur commonly, and appeared to underestimate the histological occurrence of such changes. Histological changes considered to represent CIN were uncommon in patients who said that they had begun sexual activity less than 10 years ago, whereas patients with warty characteristics on histology showed no such bias.