California Cannabis Research Briefing, April 2023: Meeting Summary.

On April 28, 2023, the University of California Office of the President, in partnership with the California Department of Cannabis Control (DCC), hosted the California Cannabis Research Briefing. The California Cannabis Research Briefing brought together researchers and state agencies/policymakers to discuss pertinent policy issues on cannabis within the state. Researchers across six different topic areas (environment, cannabis markets, social equity matters, public health, medicinal cannabis use, and public safety) provided brief explanations of their research and its policy implications.

Sequence modification on demand: search and replace tools for precise gene editing in plants.

Until recently, our ability to generate allelic diversity in plants was limited to introduction of variants from domesticated and wild species by breeding via uncontrolled recombination or the use of chemical and physical mutagens-processes that are lengthy and costly or lack specificity, respectively. Gene editing provides a faster and more precise way to create new variation, although its application in plants has been dominated by the creation of short insertion and deletion mutations leading to loss of gene function, mostly due to the dependence of editing outcomes on DNA repair pathway choices intrinsic to higher eukaryotes. Other types of edits such as point mutations and precise and pre-designed targeted sequence insertions have rarely been implemented, despite providing means to modulate the expression of target genes or to engineer the function and stability of their protein products.

Genome Editing to Achieve the Crop Ideotype in Tomato.

For centuries, combining useful traits into a single tomato plant has been done by selective crossbreeding that resulted in hundreds of extant modern cultivars. However, crossbreeding is a labor-intensive process that requires between 5 and 7 years to develop a new variety. More recently, genome editing with the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has been established as an efficient method to accelerate the breeding process by introducing targeted modifications to plant genomes via generation of targeted double-strand breaks (DSBs).

De novo domestication of wild tomato using genome editing.

Breeding of crops over millennia for yield and productivity has led to reduced genetic diversity. As a result, beneficial traits of wild species, such as disease resistance and stress tolerance, have been lost. We devised a CRISPR-Cas9 genome engineering strategy to combine agronomically desirable traits with useful traits present in wild lines.

Agrobacterium rhizogenes-mediated transformation of a dioecious plant model Silene latifolia.

Silene latifolia serves as a model species to study dioecy, the evolution of sex chromosomes, dosage compensation and sex-determination systems in plants. Currently, no protocol for genetic transformation is available for this species, mainly because S. latifolia is considered recalcitrant to in vitro regeneration and infection with Agrobacterium tumefaciens.

Novel alleles of rice eIF4G generated by CRISPR/Cas9-targeted mutagenesis confer resistance to Rice tungro spherical virus.

Rice tungro disease (RTD) is a serious constraint in rice production across tropical Asia. RTD is caused by the interaction between Rice tungro spherical virus (RTSV) and Rice tungro bacilliform virus. RTSV resistance found in traditional cultivars has contributed to a reduction in the incidence of RTD in the field.

Allele exchange at the EPSPS locus confers glyphosate tolerance in cassava.

Effective weed control can protect yields of cassava (Manihot esculenta) storage roots. Farmers could benefit from using herbicide with a tolerant cultivar. We applied traditional transgenesis and gene editing to generate robust glyphosate tolerance in cassava.

CRISPR/Cas9 and TALENs generate heritable mutations for genes involved in small RNA processing of Glycine max and Medicago truncatula.

Processing of double-stranded RNA precursors into small RNAs is an essential regulator of gene expression in plant development and stress response. Small RNA processing requires the combined activity of a functionally diverse group of molecular components. However, in most of the plant species, there are insufficient mutant resources to functionally characterize each encoding gene.

Design and Assembly of CRISPR/Cas9 Reagents for Gene Knockout, Targeted Insertion, and Replacement in Wheat.

Advances in cereal transformation along with the completion of the wheat genome sequence assembly have increased the demand for tools that perform targeted and specific modifications in this crop plant. This protocol demonstrates the construction of reagents using a comprehensive genome engineering kit to create single and multiple gene "knockouts," site-specific chromosome deletions and gene replacement or "knockins" including the use of geminivirus replicons (GVRs). The reagents allow for both easy construction of simple genome engineering vectors, and "mix and match" swapping of components such as the Cas9, guide RNA and donor template cassettes for gene targeting.

A Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants.

We report a comprehensive toolkit that enables targeted, specific modification of monocot and dicot genomes using a variety of genome engineering approaches. Our reagents, based on transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, are systematized for fast, modular cloning and accommodate diverse regulatory sequences to drive reagent expression. Vectors are optimized to create either single or multiple gene knockouts and large chromosomal deletions.

Genome editing as a tool to achieve the crop ideotype and de novo domestication of wild relatives: Case study in tomato.

The ideotype is a theoretical model of an archetypal cultivated plant. Recent progress in genome editing is aiding the pursuit of this ideal in crop breeding. Breeding is relatively straightforward when the traits in question are monogenic in nature and show Mendelian inheritance.

Fatty acid profile in erythrocyte membranes and plasma phospholipids affects significantly the extent of inflammatory response to coronary stent implantation.

In coronary heart disease, the treatment of significant stenosis by percutaneous coronary intervention (PCI) with stent implantation elicits local and systemic inflammatory responses. This study was aimed at evaluation of the dynamics of inflammatory response and elucidation of the relationship between the fatty acid profile of red blood cell (RBC) membranes or plasma phospholipids and inflammation after PCI. High-sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), serum amyloid A (SAA), malondialdehyde (MDA) and the fatty acid profiles were determined in patients with advanced coronary artery disease undergoing PCI before, 24 h and 48 h after drug-eluting stent implantation (n=36).

Prevention of Youthful Marijuana Use.

Considerable money and effort have been expended in attempts to prevent drug use by youth, with disappointing results. Too often, prevention programs have singled out youth with simplistic messages of exaggerated risk and the same politically acceptable solution for all-abstinence. Historically, prevention efforts have been less effective by not being soundly based in science and failing also to address adult drug and alcohol use as part of the problem.

Clinical Approach to the Heavy Cannabis User in the Age of Medical Marijuana.

This article begins with a case vignette exemplifying the common clinical problem of heavy marijuana users. The epidemiology and basic science underlying cannabis dependence is outlined, followed by clinical strategies for basing a therapeutic alliance on known research findings and using motivational interviewing to deal with typical patterns of denial.

A Defect in DNA Ligase4 Enhances the Frequency of TALEN-Mediated Targeted Mutagenesis in Rice.

We have established methods for site-directed mutagenesis via transcription activator-like effector nucleases (TALENs) in the endogenous rice (Oryza sativa) waxy gene and demonstrated stable inheritance of TALEN-induced somatic mutations to the progeny. To analyze the role of classical nonhomologous end joining (cNHEJ) and alternative nonhomologous end joining (altNHEJ) pathways in TALEN-induced mutagenesis in plant cells, we investigated whether a lack of DNA Ligase4 (Lig4) affects the kinetics of TALEN-induced double-strand break repair in rice cells. Deep-sequencing analysis revealed that the frequency of all types of mutations, namely deletion, insertion, combination of insertion with deletion, and substitution, in lig4 null mutant calli was higher than that in a lig4 heterozygous mutant or the wild type.

High-frequency, precise modification of the tomato genome.

Background: The use of homologous recombination to precisely modify plant genomes has been challenging, due to the lack of efficient methods for delivering DNA repair templates to plant cells. Even with the advent of sequence-specific nucleases, which stimulate homologous recombination at predefined genomic sites by creating targeted DNA double-strand breaks, there are only a handful of studies that report precise editing of endogenous genes in crop plants. More efficient methods are needed to modify plant genomes through homologous recombination, ideally without randomly integrating foreign DNA.

Efficient design and assembly of custom TALENs using the Golden Gate platform.

An important breakthrough in the field of genome engineering was the discovery of the modular Transcription Activator-Like Effector (TALE) DNA binding domain and the development of TALE nucleases (TALENs). TALENs enable researchers to make DNA double-strand breaks in target loci to create gene knockouts or introduce specific DNA sequence modifications. Precise genome engineering is increasingly being used to study gene function, develop disease models or create new traits in crop species.

Autism Spectrum Disorder: Defining Dimensions and Subgroups.

Autism spectrum disorder (ASD) is a behaviorally defined neurodevelopmental disorder associated with the presence of social-communication deficits and restricted and repetitive behaviors. In the latest conceptualization of ASD, these two behavioral dimensions represent the core defining features of ASD, whereas associated dimensions, such as intellectual and language ability, provide a means for describing the ASD heterogeneity. In addition, the characterization of ASD subgroups, defined by the presence of known medical, genetic, or other psychiatric disorders, furthers our understanding of ASD heterogeneity.

PNPLA3 variant I148M is associated with altered hepatic lipid composition in humans.

Aims/hypothesis: The common sequence variant I148M of the patatin-like phospholipase domain-containing protein 3 gene (PNPLA3) is associated with increased hepatic triacylglycerol (TAG) content, but not with insulin resistance, in humans. The PNPLA3 (I148M) variant was previously reported to alter the specificity of the encoded enzyme and subsequently affect lipid composition.

Methods: We analysed the fatty acid composition of five lipid fractions from liver tissue samples from 52 individuals, including 19 carriers of the minor PNPLA3 (I148M) variant.

Mouse genome engineering using designer nucleases.

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well as for modeling human diseases and testing therapeutic strategies. Recent advances in the use of designer nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system for site-specific genome engineering open the possibility to perform rapid targeted genome modification in virtually any laboratory species without the need to rely on embryonic stem (ES) cell technology. A genome editing experiment typically starts with identification of designer nuclease target sites within a gene of interest followed by construction of custom DNA-binding domains to direct nuclease activity to the investigator-defined genomic locus.

Possible mechanisms responsible for absence of a retrotransposon family on a plant Y chromosome.

Some transposable elements (TEs) show extraordinary variance in abundance along sex chromosomes but the mechanisms responsible for this variance are unknown. Here, we studied Ogre long terminal repeat (LTR) retrotransposons in Silene latifolia, a dioecious plant with evolutionarily young heteromorphic sex chromosomes. Ogre elements are ubiquitous in the S.

DNA replicons for plant genome engineering.

Sequence-specific nucleases enable facile editing of higher eukaryotic genomic DNA; however, targeted modification of plant genomes remains challenging due to ineffective methods for delivering reagents for genome engineering to plant cells. Here, we use geminivirus-based replicons for transient expression of sequence-specific nucleases (zinc-finger nucleases, transcription activator-like effector nucleases, and the clustered, regularly interspaced, short palindromic repeat/Cas system) and delivery of DNA repair templates. In tobacco (Nicotiana tabacum), replicons based on the bean yellow dwarf virus enhanced gene targeting frequencies one to two orders of magnitude over conventional Agrobacterium tumefaciens T-DNA.

Isolated peritoneal donor-related plasmacytoma 3 years after liver transplantation: a case report.

Organ transplantation carries a risk of disease transmission from donor to recipient, primarily infection or malignancy. Although donors are thoroughly screened, donor-related malignancies are reported to occur in 0.01% of solid organ transplants.

Does on-site adequacy evaluation reduce the nondiagnostic rate in endoscopic ultrasound-guided fine-needle aspiration of pancreatic lesions?

Background: This retrospective study compared the nondiagnostic rate for endoscopic ultrasound-guided (EUS) fine-needle aspiration (FNA) of pancreatic lesions in 2 settings: 1 with and 1 without on-site evaluation.

Methods: The authors reviewed 381 consecutive cases and divided them into groups with and without on-site adequacy evaluation. For the group with on-site evaluation, cytopathology personnel prepared and evaluated Diff-Quik-stained direct smears and rinsed the remaining material in CytoLyt solution (Cytyc Corporation, Marlborough, Mass).