Biology subjects

Howell, A. L.

Publications and source records attributed to Howell, A. L..

4 recordsLinked to original sources

bioRxiv2025

Enhanced cleavage of genomic CCR5 using CASX2Max

Development of novel CRISPR/Cas systems enhances opportunities for gene editing to treat infectious diseases, cancer, and genetic disorders. We evaluated CasX2 (PlmCas12e), a class II CRISPR system derived from Planctomycetes, a non-pathogenic bacterium present in aquatic and terrestrial soils. CasX2 offers several advantages over Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus aureus Cas9 (SaCas9), including its smaller size, distinct protospacer adjacent motif (PAM) requirements, staggered cleavage cuts that promote homology-directed repair, and no known pre-existing immunity in humans. A recent study reported that a three amino acid substitution in CasX2 significantly enhanced cleavage activity (1). Therefore, we compared cleavage efficiency and double-stranded break repair characteristics between the native CasX2 and the variant, CasX2Max, for cleavage of CCR5, a gene that encodes the CCR5 receptor important for HIV-1 infection. Two CasX2 single guide RNAs (sgRNAs) were designed that flanked the 32 bases deleted in the natural CCR5 {Delta}32 mutation. Nanopore sequencing demonstrated that CasX2 using sgRNAs with spacers of 17 nucleotides (nt), 20 nt or 23 nt in length were ineffective at cleaving genomic CCR5. In contrast, CasX2Max using sgRNAs with 20 nt and 23 nt spacer lengths, enabled robust genomic cleavage of CCR5. Structural modeling indicated that two of the CasX2Max substitutions enhanced sgRNA-DNA duplex stability, while the third improved DNA strand alignment within the catalytic site. These structural changes likely underlie the increased activity of CasX2Max in cellular gene excision. In sum, CasX2Max consistently outperformed native CasX2 across all assays and represents a superior gene-editing platform for therapeutic applications.

molecular biology↗

bioRxiv2025

Optimizing Protocols for MicroRNA Profiling of Infant and Toddler Stool

BackgroundMicroRNAs (miRNAs) are increasingly being investigated as potential biomarkers for child development and disease. Although a growing number of studies are utilizing infant and toddler stool for transcriptomic analyses, no studies have compared protocols for preserving and extracting miRNAs from this specimen type, despite unique challenges, including abundant levels of RNAses and microbial RNA. MethodsTo address this, we first compared three commercially available kits and four preservation methods for their ability to yield high quality RNA from infant and toddler stool (Phase 1). RNA quality was determined by fragment analyzer. ResultsOf the three RNA extraction kits compared, Zymo BIOMICs yielded the highest overall RNA Quality Number (RQN) (median (range) RQN 9.4 (5.7-10.0)). Of the four preservation methods tested, stool collected in RNAlater and Zymo DNA/RNA Shield Fecal Collection Tubes yielded the highest two RQNs (median (range) RQN 9.8 (5.7-10.0) and 9.4 (5.4-10.0), respectively), which did not differ significantly from each other (p = 0.47). Second, using miRNA-seq we directly compared miRNA profiles for RNA extracted using the Zymo BIOMICs kit from paired aliquots of the same stool sample from four infants collected into RNAlater and Zymo DNA/RNA Shield Fecal Collection Tubes (Phase 2). Given that microbial sequences greatly outnumber human miRNAs in stool, reads were first classified as human versus microbial prior to aligning human-classified reads to miRBase v22.1. The percentage of reads classified as human and the percentage of human reads aligning to miRBase did not differ for samples collected in RNAlater versus Zymo Shield (p = 0.12 and p = 0.86, respectively). Furthermore, after multiple testing correction, normalized miRNA counts did not differ significantly between the two preservatives for any of the 42 human miRNAs detected across the eight samples. ConclusionsCollecting infant and toddler stool in either RNAlater or Zymo DNA/RNA Shield Fecal Collection Tubes, when paired with RNA extraction using the Zymo BIOMICs extraction kit, yielded high-quality RNA with similar human miRNA profiles. Moreover, of the 42 miRNAs that were detected, several (i.e., miR-194a-3p, miR-200c-3p, miR-26a-5p) are thought to contribute to overall gut homeostasis. These findings may inform protocols for future studies that aim to profile miRNAs in infant and toddler stool to evaluate their potential utility as biomarkers for childrens health.

developmental biology↗

bioRxiv2023

CAS12e (CASX2) CLEAVAGE OF CCR5: IMPACT OF GUIDE RNA LENGTH AND PAM SEQUENCE ON CLEAVAGE ACTIVITY

CRISPR/Cas is under development as a therapeutic tool for the cleavage, excision, and/or modification of genes in eukaryotic cells. While much effort has focused on CRISPR/Cas from Streptococcus pyogenes (SpCas9) and Staphylococcus aureus (SaCas9), alternative CRISPR systems have been identified using metagenomic datasets from non-pathogenic microbes, including previously unknown class 2 systems, adding to a diverse toolbox of gene editors. The Cas12e (CasX1, CasX2) endonucleases from non-pathogenic Deltaproteobacteria (DpeCas12e) and Planctomycetes (PlmCas12e) are more compact than SpCas9, have a more selective protospacer adjacent motif (PAM) requirement, and deliver a staggered cleavage cut with 5-7 base overhangs. We investigated varying guide RNA (spacer) lengths and alternative PAM sequences to determine optimal conditions for PlmCas12e cleavage of the cellular gene CCR5 (CC-Chemokine receptor-5). CCR5 encodes one of two chemokine coreceptors required by HIV-1 to infect target cells, and a mutation of CCR5 (delta-32) is responsible for HIV-1 resistance and reported cures following bone marrow transplantation. Consequently, CCR5 has been an important target for gene editing utilizing CRISPR, TALENs, and ZFNs. We determined that CCR5 cleavage activity varied with the target site, guide RNA length, and the terminal nucleotide in the PAM sequence. Our analyses demonstrated a PlmCas12e PAM preference for purines (A, G) over pyrimidines (T, C) in the fourth position of the CasX2 PAM (TTCN). These analyses have contributed to a better understanding of CasX2 cleavage requirements and will position us more favorably to develop a therapeutic that creates the delta-32 mutation in the CCR5 gene in hematopoietic stem cells.

molecular biology↗

bioRxiv2019

The intrinsic neonatal hippocampal network: rsfMRI findings

Many adults cannot voluntarily recall memories before the ages of 3-5, a phenomenon referred to as "infantile amnesia" The development of the hippocampal network likely plays a significant part in the emergence of the ability to form long-lasting memories. In adults, the hippocampus has specialized and privileged connections with certain cortical networks, which presumably facilitate its involvement in memory encoding, consolidation, and retrieval. Is the hippocampus already specialized in these cortical connections at birth? And are the topographical principles of connectivity (e.g. long-axis specialization) present at birth? We analyzed resting-state hippocampal connectivity in neonates scanned within one week of birth (Developmental Human Connectome Project) and compared them to adults (Human Connectome Project). We explored the connections of the whole hippocampus and its long-axis specialization to seven canonical cortical networks. We found that the neonatal hippocampal networks show clear immaturity at birth: adults showed hippocampal connectivity that was unique for each cortical network, whereas neonates showed no differentiation in hippocampal connectivity across these networks. Further, neonates lacked long-axis specialization (i.e., along anterior-posterior axis) of the hippocampus in its differential connectivity patterns to the cortical networks. This immaturity in connectivity may contribute to immaturity in memory formation in the first years of life. "New and Noteworthy"While animal data, and anatomical and behavioral human data from young children suggest that the hippocampus is immature at birth, to date, there are no direct assessments of human hippocampal functional connectivity (FC) very early in life. Our study explores the FC of the hippocampus to the cortex at birth, allowing insight into the development of human memory systems.

neuroscience↗