CRISPR/Cas9 deletions induce adverse on-target genomic effects leading to functional DNA in human cells

The CRISPR/Cas9 system is widely used to permanently delete genomic regions by inducing double-strand breaks via dual guide RNAs. However, consequences of Cas9 deletion events have not been fully investigated. To characterize Cas9-induced genotypic abnormalities in human cells, we utilized an innovative droplet-based target enrichment approach followed by long-read sequencing and coupled it to customized de novo sequence assembly. We here describe extensive genomic disruptions by Cas9, involving a genomic duplication and inversion of the target region as well as integrations of exogenous DNA at the double-strand break sites. Although these events altered the genomic composition of the on-target region, we found that the aberrant DNA fragments are still functional, marked by active histones and bound by RNA polymerase III. Our findings broaden the consequential spectrum of the Cas9 deletion system, reinforce the necessity of meticulous genomic validations and rationalize extra caution when interpreting results from a deletion event.

CMV, MHC-E, and the quest for an unconventional AIDS vaccine

In the evolutionary battle between virus and host, a genetic alteration in cytomegalovirus caused by an inversion-deletion event during tissue culture passage opens an unconventional path toward an HIV vaccine (see the related Research Articles by Malouli et al., Yang et al., and Verweij et al.).

Genomic Characterization of Salmonella typhimurium DT104 Strains Associated with Cattle and Beef Products

Salmonella enterica subsp. enterica serovar Typhimurium DT104, a multidrug-resistant phage type, has emerged globally as a major cause of foodborne outbreaks particularly associated with contaminated beef products. In this study, we sequenced three S. Typhimurium DT104 strains associated with a 2009 outbreak caused by ground beef, including the outbreak source strain and two clinical strains. The goal of the study was to gain a stronger understanding of the genomics and genomic epidemiology of highly clonal S. typhimurium DT104 strains associated with bovine sources. Our study found no single nucleotide polymorphisms (SNPs) between the ground beef source strain and the clinical isolates from the 2009 outbreak. SNP analysis including twelve other S. typhimurium strains from bovine and clinical sources, including both DT104 and non-DT104, determined DT104 strains averaged 55.0 SNPs between strains compared to 474.5 SNPs among non-DT104 strains. Phylogenetic analysis separated the DT104 strains from the non-DT104 strains, but strains did not cluster together based on source of isolation even within the DT104 phage type. Pangenome analysis of the strains confirmed previous studies showing that DT104 strains are missing the genes for the allantoin utilization pathway, but this study confirmed that the genes were part of a deletion event and not substituted or disrupted by the insertion of another genomic element. Additionally, cgMLST analysis revealed that DT104 strains with cattle as the source of isolation were quite diverse as a group and did not cluster together, even among strains from the same country. Expansion of the analysis to 775 S. typhimurium ST19 strains associated with cattle from North America revealed diversity between strains, not limited to just among DT104 strains, which suggests that the cattle environment is favorable for a diverse group of S. typhimurium strains and not just DT104 strains.

Sequence Read Depth Analysis of a Monophyletic Cluster of Y Chromosomes Characterized by Structural Rearrangements in the AZFc Region Resulting in DYS448 Deletion and DYF387S1 Duplication

The azoospermia factor c region (AZFc), located in the long arm of the human Y chromosome, is frequently involved in chromosome rearrangements, mainly due to non-allelic homologous recombination events that occur between the nearly identical sequences (amplicon) that comprises it. These rearrangements may have major phenotypic effects like spermatogenic failure or other pathologies linked to male infertility. Moreover, they may also be relevant in forensic genetics, since some of the Y chromosome short tandem repeats (Y-STRs) commonly used in forensic analysis are located in amplicons or in inter-amplicon sequences of the AZFc. In a previous study, we identified four phylogenetically related samples with a null allele at DYS448 and a tetrallelic pattern at DYF387S1, two Y-STRs located in the AZFc. Through NGS read depth analysis, we found that the unusual Y-STR pattern may be due to a 1.6 Mb deletion arising concurrently or after a 3.5 Mb duplication event. The observed large genomic rearrangement results in copy number reduction for the RBMY gene family as well as duplication of other AZFc genes. Based on the diversity of 16 additional Y-STRs, we estimated that the duplication/deletion event occurred at least twenty generations ago, suggesting that it has not been affected by negative selection.

Analysis of a small outbreak of Shiga toxin-producing Escherichia coli O157:H7 using long-read sequencing

Compared to short-read sequencing data, long-read sequencing facilitates single contiguous de novo assemblies and characterization of the prophage region of the genome. Here, we describe our methodological approach to using Oxford Nanopore Technology (ONT) sequencing data to quantify genetic relatedness and to look for microevolutionary events in the core and accessory genomes to assess the within-outbreak variation of four genetically and epidemiologically linked isolates. Analysis of both Illumina and ONT sequencing data detected one SNP between the four sequences of the outbreak isolates. The variant calling procedure highlighted the importance of masking homologous sequences in the reference genome regardless of the sequencing technology used. Variant calling also highlighted the systemic errors in ONT base-calling and ambiguous mapping of Illumina reads that results in variations in the genetic distance when comparing one technology to the other. The prophage component of the outbreak strain was analysed, and nine of the 16 prophages showed some similarity to the prophage in the Sakai reference genome, including the stx2a-encoding phage. Prophage comparison between the outbreak isolates identified minor genome rearrangements in one of the isolates, including an inversion and a deletion event. The ability to characterize the accessory genome in this way is the first step to understanding the significance of these microevolutionary events and their impact on the evolutionary history, virulence and potentially the likely source and transmission of this zoonotic, foodborne pathogen.

An Indian diagnostic laboratory case report on mosaic chromosome 18

<p class="abstract">Distal 18q deletion syndrome, and ring chromosome 18 are structural abnormalities involving chromosome 18. Distal or terminal deletion event is characterized by deletion of a region from the terminal end of a chromosome, while in ring chromosome, the telomeric ends of both the arms of homologous chromosomes are lost, causing the sticky ends to fuse together to form a ring. Clinical findings vary in each case depending on the type of abnormality, and the region of chromosome affected. This case report focuses on the chromosome 18 abnormality detected in the proband tested with the clinical indication of cleft lip, congenital anomaly, and feeding difficulty. Parental karyotyping to rule out de-novo or abnormality due to inheritance, clinical correlation, genetic counseling and high resolution microarray was recommended.</p>

Metabolic selection of a homologous recombination mediated loss of glycosomal fumarate reductase in Trypanosoma brucei

The genome of trypanosomatids is rearranged at the level of repeated sequences, where serve as platforms for amplification or deletion of genomic segments. We report here that the PEPCK gene knockout (Δpepck) leads to the selection of such a deletion event between the FRDg and FRDm2 genes to produce a chimeric FRDg-m2 gene in the Δpepck* cell line. FRDg is expressed in peroxisome-like organelles, named glycosomes, expression of FRDm2 has not been detected to date, and FRDg-m2 is a non-functional cytosolic FRD. Re-expression of FRDg significantly impaired growth of the Δpepck* cells, while inhibition of FRDg-m2 expression had no effect, which indicated that this recombination event has been selected in the Δpepck* cells to eliminate FRDg. FRD activity was not involved in the FRDg-mediated negative effect, while its auto-flavinylation motif is required to impair growth. Considering that (i) FRDs are known to generate reactive oxygen species (ROS) by transferring electrons from their flavin moiety(ies) to oxygen, (ii) intracellular ROS production is essential for the differentiation of procyclic to epimastigote forms of the parasite and (iii) the fumarate reductase activity is not essential for the parasite, we propose that the main role of FRD is to produce part of the ROS necessary to complete the parasitic cycle in the tsetse fly. In this context, the negative effect of FRDg expression in the PEPCK null background is interpreted as an increased production of ROS from oxygen since fumarate, the natural electron acceptor of FRDg, is no longer produced in glycosomes.

Formation of ultralong DH regions through genomic rearrangement

Background Cow antibodies are very unusual in having exceptionally long CDR H3 regions. The genetic basis for this length largely derives from long heavy chain diversity (DH) regions, with a single “ultralong” DH, IGHD8–2, encoding over 50 amino acids. Many bovine IGHD regions have sequence similarity but have several nucleotide repeating units that diversify their lengths. Genomically, most DH regions exist in three clusters that appear to have formed from DNA duplication events. However, the relationship between the genomic arrangement and long CDR lengths is unclear. Results The DH cluster containing IGHD8–2 underwent a rearrangement and deletion event in relation to the other clusters in the region corresponding to IGHD8–2, with possible fusion of two DH regions and expansion of short repeats to form the ultralong IGHD8–2 gene. Conclusions Length heterogeneity within DH regions is a unique evolutionary genomic mechanism to create immune diversity, including formation of ultralong CDR H3 regions.

Formation of ultralong DH regions through genomic rearrangement

Background: Cow antibodies are very unusual in having exceptionally long CDR H3 regions. The genetic basis for this length largely derives from long heavy chain diversity (DH) regions, with a single “ultralong” DH, IGHD8-2, encoding over fifty amino acids. Many bovine IGHD regions have sequence similarity but have several nucleotide repeating units that diversify their lengths. Genomically, most DH regions exist in three clusters that appear to have formed from DNA duplication events. However, the relationship between the genomic arrangement and long CDR lengths is unclear. Results: The DH cluster containing IGHD8-2 underwent a rearrangement and deletion event in relation to the other clusters in the region corresponding to IGHD8-2, with possible fusion of two DH regions and expansion of short repeats to form the ultralong IGHD8-2 gene. Conclusions: Length heterogeneity within DH regions is a unique evolutionary genomic mechanism to create immune diversity, including formation of ultralong CDR H3 regions.

Type I-F CRISPR-Cas Distribution and Array Dynamics in Legionella pneumophila

In bacteria and archaea, several distinct types of CRISPR-Cas systems provide adaptive immunity through broadly similar mechanisms: short nucleic acid sequences derived from foreign DNA, known as spacers, engage in complementary base pairing with invasive genetic elements setting the stage for nucleases to degrade the target DNA. A hallmark of type I CRISPR-Cas systems is their ability to acquire spacers in response to both new and previously encountered invaders (naïve and primed acquisition, respectively). Our phylogenetic analyses of 43 L. pneumophila type I-F CRISPR-Cas systems and their resident genomes suggest that many of these systems have been horizontally acquired. These systems are frequently encoded on plasmids and can co-occur with nearly identical chromosomal loci. We show that two such co-occurring systems are highly protective and undergo efficient primed acquisition in the lab. Furthermore, we observe that targeting by one system’s array can prime spacer acquisition in the other. Lastly, we provide experimental and genomic evidence for a model in which primed acquisition can efficiently replenish a depleted type I CRISPR array following a mass spacer deletion event.