Long-range Deletions Research Articles

Targeted large fragment deletion in plants using paired crRNAs with type I CRISPR system.

The CRISPR-Cas systems have been widely used as genome editing tools, with type II and V systems typically introducing small indels, and type I system mediating long-range deletions. However, the precision of type I systems for large fragment deletion is still remained to be optimized. Here, we developed a compact Cascade-Cas3 Dvu I-C system with Cas11c for plant genome editing. The Dvu I-C system was efficient to introduce controllable large fragment deletion up to at least 20 kb using paired crRNAs. The paired-crRNAs design also improved the controllability of deletions for the type I-E system. Dvu I-C system was sensitive to spacer length and mismatch, which was benefit for target specificity. In addition, we showed that the Dvu I-C system was efficient for generating stable transgenic lines in maize and rice with the editing efficiency up to 86.67%. Overall, Dvu I-C system we developed here is powerful for achieving controllable large fragment deletions.

Repurposing the atypical type I-G CRISPR system for bacterial genome engineering.

The CRISPR-Cas system functions as a prokaryotic immune system and is highly diverse, with six major types and numerous sub-types. The most abundant are type I CRISPR systems, which utilize a multi-subunit effector, Cascade, and a CRISPR RNA (crRNA) to detect invading DNA species. Detection leads to DNA loading of the Cas3 helicase-nuclease, leading to long-range deletions in the targeted DNA, thus providing immunity against mobile genetic elements (MGE). Here, we focus on the type I-G system, a streamlined, 4-subunit complex with an atypical Cas3 enzyme. We demonstrate that Cas3 helicase activity is not essential for immunity against MGE in vivo and explore applications of the Thioalkalivibrio sulfidiphilus Cascade effector for genome engineering in Escherichia coli. Long-range, bidirectional deletions were observed when the lacZ gene was targeted. Deactivation of the Cas3 helicase activity dramatically altered the types of deletions observed, with small deletions flanked by direct repeats that are suggestive of microhomology mediated end joining. When donor DNA templates were present, both the wild-type and helicase-deficient systems promoted homology-directed repair (HDR), with the latter system providing improvements in editing efficiency, suggesting that a single nick in the target site may promote HDR in E. coli using the type I-G system. These findings open the way for further application of the type I-G CRISPR systems in genome engineering.

Genetic and clinical screening for hereditary primary hyperparathyroidism in a large Chinese cohort: a single-center study.

Primary hyperparathyroidism (PHPT) includes sporadic PHPT and hereditary PHPT. However, until now, there have been no exact data on the proportion and composition of hereditary PHPT in the Chinese PHPT population. This study aimed to clarify the proportion and composition of hereditary PHPT in patients at a large academic center in Beijing, China, and to analyze genotype-phenotype characteristics. A total of 394 newly diagnosed Han PHPT patients who consented to genetic screening were enrolled. Targeted next-generation sequencing (T-NGS) (including for MEN1, RET, CDKN1B, CaSR, HRPT2/CDC73, GNA11, AP2S1, GCM2), combined with MEN1-multiplex ligation-dependent probe amplification (MLPA) and CDC73-MLPA, was used for genetic screening. Diagnosis of hereditary PHPT was based on clinical manifestations, family history, and genetic screening. Thirty-seven pathogenic (P)/likely pathogenic (LP) variants were detected in 41 patients via T-NGS, and three patients carried long-range deletions of MEN1 or CDC73 detected by MLPA, with a variant detection rate of 11.2% (44/394). In total, 30 patients were clinically diagnosed with MEN1. Combined with genetic and clinical screening, the rate of hereditary PHPT in this study was 18.8% (74/394). For purposes of comparison, the rate of unequivocal nonhereditary PHPT was 66.5% (262/394); 14.7% (58/394) did not exhibit the clinical features of hereditary PHPT but carried variants of uncertain clinical significance and so could not be clearly categorized. Both the age at hospital visit (43.6 ± 14.0 versus 53.7 ± 14.9 years) and age at onset (35.4 ± 13.8 versus 50.6 ± 14.8 years) in the hereditary group (n = 74) were significantly lower than those in the nonhereditary group (n = 262). Higher levels of ionized calcium and serum β-CTX were observed in the hereditary group; proportions of parathyroid hyperplasia and multigland involvement were also higher. In addition to multigland disease and positive family history, it is recommended that patients with an age of onset less than 38 should be screened for hereditary forms. © 2023 American Society for Bone and Mineral Research (ASBMR).

Type I-D CRISPR System-Mediated Genome Editing in Plants.

Genome editing has revolutionized plant research and plant breeding by enabling precise genome manipulation. In particular, the application of type II CRISPR-Cas9 systems to genome editing has proved an important milestone, accelerating genetic engineering and the analysis of gene function. On the other hand, the potential of other types of CRISPR-Cas systems, especially many of the most abundant type I CRISPR-Cas systems, remains unexplored. We recently developed a novel genome editing tool, TiD, based on the type I-D CRISPR-Cas system. In this chapter, we describe a protocol for genome editing of plant cells using TiD. This protocol allows the application of TiD to induce short insertion and deletions (indels) or long-range deletions at target sites with high specificity in tomato cells.

Expanding the plant genome editing toolbox with recently developed CRISPR-Cas systems.

Since its first appearance, CRISPR–Cas9 has been developed extensively as a programmable genome-editing tool, opening a new era in plant genome engineering. However, CRISPR–Cas9 still has some drawbacks, such as limitations of the protospacer-adjacent motif (PAM) sequence, target specificity, and the large size of the cas9 gene. To combat invading bacterial phages and plasmid DNAs, bacteria and archaea have diverse and unexplored CRISPR–Cas systems, which have the potential to be developed as a useful genome editing tools. Recently, discovery and characterization of additional CRISPR–Cas systems have been reported. Among them, several CRISPR–Cas systems have been applied successfully to plant and human genome editing. For example, several groups have achieved genome editing using CRISPR–Cas type I-D and type I-E systems, which had never been applied for genome editing previously. In addition to higher specificity and recognition of different PAM sequences, recently developed CRISPR–Cas systems often provide unique characteristics that differ from well-known Cas proteins such as Cas9 and Cas12a. For example, type I CRISPR–Cas10 induces small indels and bi-directional long-range deletions ranging up to 7.2 kb in tomatoes (Solanum lycopersicum L.). Type IV CRISPR–Cas13 targets RNA, not double-strand DNA, enabling highly specific knockdown of target genes. In this article, we review the development of CRISPR–Cas systems, focusing especially on their application to plant genome engineering. Recent CRISPR–Cas tools are helping expand our plant genome engineering toolbox.

A novel long-range deletion spanning CDC73 and upper-stream genes discovered in a kindred of familial primary hyperparathyroidism.

To confirm the exact break-point of a novel long-range deletion discovered in one female parathyroid carcinoma (PC) patient who has a strong family history suggesting familial hyperparathyroidism, and to investigate the expression of parafibromin in the patient's affected lesion. Clinical information of one female patient as well as five of her relatives was collected. Their genomic DNA extracted from peripheral blood went through Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). After completing whole genome sequencing (WGS), clone sequencing was also performed, whose result was aligned with standard human genome database after Sanger sequencing. The medical history of recurrent hypercalcemia after parathyroidectomy and histopathological investigation confirmed that the female patient was diagnosed with PC. WGS displayed a novel 130 kb long-range deletion spanning UCHL5 to CDC73 that was later confirmed by clone sequencing. MLPA showed similar results in four of her five relatives, suggesting these people to be carriers of the same long-range deletion, and three among them had a history of primary hyperparathyroidism (PHPT) ahead of the proband's first visit. We discovered a novel 130 kb long-range deletion spanning CDC73 in a family of 5 persons, and the existence of the deletion was related to PHPT and PC. Our discovery validated the role of CDC73 mutation in the occurrence of PHPT and PC, which provided new information to the genetic studies of PC.

Genome editing in mammalian cells using the CRISPR type I-D nuclease.

Adoption of CRISPR–Cas systems, such as CRISPR–Cas9 and CRISPR–Cas12a, has revolutionized genome engineering in recent years; however, application of genome editing with CRISPR type I—the most abundant CRISPR system in bacteria—remains less developed. Type I systems, such as type I-E, and I-F, comprise the CRISPR-associated complex for antiviral defense (‘Cascade’: Cas5, Cas6, Cas7, Cas8 and the small subunit) and Cas3, which degrades the target DNA; in contrast, for the sub-type CRISPR–Cas type I-D, which lacks a typical Cas3 nuclease in its CRISPR locus, the mechanism of target DNA degradation remains unknown. Here, we found that Cas10d is a functional nuclease in the type I-D system, performing the role played by Cas3 in other CRISPR–Cas type I systems. The type I-D system can be used for targeted mutagenesis of genomic DNA in human cells, directing both bi-directional long-range deletions and short insertions/deletions. Our findings suggest the CRISPR–Cas type I-D system as a unique effector pathway in CRISPR that can be repurposed for genome engineering in eukaryotic cells.

Genome editing in plants using CRISPR type I-D nuclease

Genome editing in plants has advanced greatly by applying the clustered regularly interspaced short palindromic repeats (CRISPRs)-Cas system, especially CRISPR-Cas9. However, CRISPR type I—the most abundant CRISPR system in bacteria—has not been exploited for plant genome modification. In type I CRISPR-Cas systems, e.g., type I-E, Cas3 nucleases degrade the target DNA in mammals. Here, we present a type I-D (TiD) CRISPR-Cas genome editing system in plants. TiD lacks the Cas3 nuclease domain; instead, Cas10d is the functional nuclease in vivo. TiD was active in targeted mutagenesis of tomato genomic DNA. The mutations generated by TiD differed from those of CRISPR/Cas9; both bi-directional long-range deletions and short indels mutations were detected in tomato cells. Furthermore, TiD can be used to efficiently generate bi-allelic mutant plants in the first generation. These findings indicate that TiD is a unique CRISPR system that can be used for genome engineering in plants.

Deletions by type I CRISPR

Biotechnology RNA-guided recognition of target DNA by the type I CRISPR-cascade complex recruits the helicase-nuclease Cas3, which translocates along and shreds DNA in one direction. Taking advantage of this phenomenon, Dolan et al. applied Cascade-Cas3 to achieve long-range deletions in eukaryotic cells. Electroporation of the Cascade-Cas3 and guide RNA complex into human cells led to a collection of unidirectional deletions ranging from a few hundred base pairs to 100 kilobases at the target sites. This gene-editing tool might be particularly useful for creating libraries of deletions to study long cis-regulatory elements on genetic and epigenetic levels. Mol. Cell 10.1016/j.molcel.2019.03.014 (2019).

A summary of molecular genetic findings in fructose-1,6-bisphosphatase deficiency with a focus on a common long-range deletion and the role of MLPA analysis.

BackgroundFructose-1,6-bisphosphatase deficiency is a rare inborn error of metabolism affecting gluconeogenesis with only sporadic reports on its molecular genetic basis.ResultsWe report our experience with mutation analysis in 14 patients (13 families) with fructose-1,6-bisphosphatase deficiency using conventional Sanger sequencing and multiplex ligation-dependent probe amplification analysis, and we provide a mutation update for the fructose bisphosphatase-1 gene (FBP1). Mutations were found on both chromosomes in all of our 14 patients including 5 novel mutations. Among the novel mutations is a 5412-bp deletion (c.-24-26_170 + 5192del) including the entire coding sequence of exon 2 of FBP1 that was repeatedly found in patients from Turkey and Armenia which may explain earlier poorly defined findings in patients from this area. This deletion can be detected with specific primers by generation of a junction fragment and by MLPA and SNP array assays. MLPA analysis was able to detect copy number variations in two further patients, one heterozygous for a deletion within exon 8, another heterozygous for a novel deletion of the entire FBP1 gene.ConclusionsBased on our update for the FBP1 gene, currently listing 35 mutations worldwide, and knowledge of PCR conditions that allow simple detection of a common FBP1 deletion in the Armenian and Turkish population, molecular genetic diagnosis has become easier in FBP1 deficiency. Furthermore, MLPA analysis may plays a useful role in patients with this disorder.Electronic supplementary materialThe online version of this article (doi:10.1186/s13023-016-0415-1) contains supplementary material, which is available to authorized users.

Whole Genome Sequencing of Indolent CLL Reveals Novel Structural Rearrangements

Several recent next generation sequencing studies have uncovered mutations in key genes that contribute to CLL pathogenesis. Most CLLs with deletions of chromosome 13q (del(13q), incidence 50-60%) or normal cytogenetics (incidence 15-20%) do not carry these recurrent driver mutations. Clues to the genetic basis of disease in this more indolent group likely lie elsewhere in the genome.We present preliminary data from whole-genome sequencing (WGS) of a cohort of CLL patients with del(13q) (n=13) and normal cytogenetics (n=10), sequenced at 30X and 60X coverage for tumor and normal (saliva) respectively. Of the 23 patients, 43% were male and the median age at diagnosis was 53 years. The median time from diagnosis to sampling was 1.9 years, and 22 of 23 patients were previously untreated. With a median follow-up of 4.8 years in the overall cohort, 8 patients (35%) had been treated at a median 2.5 years from diagnosis. Although indolent by cytogenetics and of low Rai stage (median 1), the cohort includes balanced numbers of ZAP70± and IGHV mutated/unmutated cases.A total of 68,928 point mutations with an average of 2,995 mutations per genome were detected in our cohort using the MuTect algorithm, which is approximately 60% fewer mutations than in multiple myeloma. The pattern of mutations in protein coding regions was similar to previous exome sequencing studies, with an average of 18.7 ±7.4 non-synonymous mutations per case (0.57±0.2 per Mb). The rate of mutations was significantly lower in patients diagnosed at a younger age (p=0.016, 2 sided t test). As expected based on the cytogenetic makeup of our cohort, the incidence of mutations in the 20 known CLL driver genes did not reach genome wide significance. However, the incidence of mutations in these driver genes was significantly higher in patients with normal cytogenetics as compared to del(13q) (60% vs 18%, p=0.039).Rearrangement events were identified using the dRanger algorithm followed by mapping of the precise breakpoint to single base pair resolution using the Breakpointer algorithm. A total of 78 rearrangements were identified, corresponding to a median of 3 (range 0-15) per CLL genome, significantly fewer than in most other cancers. Similar to other hematologic malignancies, the incidence of deletions (70%) was significantly higher than that of tandem duplications (9%) (Wilcoxon signed rank test, p=0.0001). In addition, inter-chromosomal translocations exhibited a strong positive correlation with long range deletions (Pearson’s r=0.72, p=0.0001) and a moderate positive correlation with inversions (Pearson’s r=0.64, p=0.0009), suggesting a common mechanism. The only clinical feature associated with the presence of structural rearrangements was ZAP70 positivity (p=0.046).Of the 13 patients with del(13q), 4 were due to inter-chromosomal rearrangement that led to loss of the common minimal region of deletion. The other most recurrent rearrangement partners were chr.2 (n=12 cases, 52%), chr.14 (n=9 cases, 39%) and chr.1 (n=5 cases, 21%), while no rearrangements were seen in chromosomes 8,11,17,18 and 19. The remaining chromosomes were affected in <20% cases. Chr.2 events accounted for 32% of rearrangements (25/78) with breakpoints scattered throughout the chromosome and consisting of 32% inversions, 28% long-range deletions, 20% inter-chromosomal translocations, 16% deletions and 4% tandem-duplications. Chr. 2p16 gain, a recurrent chromosomal abnormality in CLL, was not detected in our cohort, and therefore not associated with these rearrangements.Chr.14 accounted for 17% of rearrangements (13/78), with breakpoints concentrated in the 5’IgH region (chr.14q32.33), in the vicinity of the KIAA0125 gene. Rearrangements involving chr.14q32.33 (n=11 total, 7 deletions) were associated with a shorter time to first therapy (TTFT) (HR=2.88, p=0.013), despite no association with IGHV or ZAP70 status.In summary, this cohort of CLLs with del(13q) and normal cytogenetics represents the largest WGS cohort reported to date and underscores the stability of the CLL genome. Nonetheless, structural rearrangements are common, and the number of non-13q rearrangements was associated with higher risk ZAP70 status. Furthermore 5’IgH rearrangements enriched in deletions were associated with shorter TTFT within this lower risk subgroup. Additional WGS data on novel somatic mutations and regulatory regions will be presented at the meeting. DisclosuresNo relevant conflicts of interest to declare.

A Role for BLM in Double-Strand Break Repair Pathway Choice: Prevention of CtIP/Mre11-Mediated Alternative Nonhomologous End-Joining

The choice of the appropriate double-strand break (DSB) repair pathway is essential for the maintenance of genomic stability. Here, we show that theBloom syndrome gene product, BLM, counteracts CtIP/MRE11-dependent long-range deletions (>200bp) generated by alternative end-joining (A-EJ). BLM represses A-EJ in an epistatic manner with 53BP1 and RIF1 and is required for ionizing-radiation-induced 53BP1 focus assembly. Conversely, in the absence of 53BP1 or RIF1, BLM promotes formation of A-EJ long deletions, consistent with a role for BLM in DSB end resection. These data highlight a dual role for BLM that influences the DSB repair pathway choice: (1) protection against CtIP/MRE11 long-range deletions associated with A-EJ and (2) promotion of DNA resection. These antagonist roles can be regulated, according to cell-cycle stage, by interacting partners such as 53BP1 and TopIII, to avoid unscheduled resection that might jeopardize genome integrity.

A protocol for construction of gene targeting vectors and generation of homologous recombinant embryonic stem cells.

The completion of human and mouse genome sequencing has confronted us with huge amount of data sequences that certainly need decades and many generations of scientists to be reasonably interpreted and assigned to physiological functions, and subsequently fruitfully translated into medical application. A means to assess the function of genes provides gene targeting in mouse embryonic stem cells (ESCs) that enables to introduce site-specific modifications in the mouse genome, and analyze their physiological consequences. Gene targeting enables almost any type of genetic modifications of interest, ranging from gene insertion (e.g., insertion of human-specific genes or reporter genes), gene disruption, point mutations, and short- and long-range deletions, inversions. Site-specific modification into the genome of ESCs can be reached by homologous recombination using targeting vectors. Here, we describe a protocol to generate targeting constructs and homologous recombinant ESCs.

A Mammary-Specific, Long-range Deletion on Mouse Chromosome 11 Accelerates Brca1-Associated Mammary Tumorigenesis

We engineered a mammary-specific knockout model for Brca1 deficiency that also lacks the majority of one chromosome 11 to determine whether tumor susceptibility loci reside on this chromosome that cooperate with the loss of Brca1 during mammary cancer formation. Brca1-deficient females that are haploinsufficient in 60 cM of chromosome 11 exhibited accelerated mammary tumorigenesis in comparison to Brca1 conditional knockout mice. On the histopathologic level, these tumors were either adenocarcinomas or benign, inflammatory lesions. Like human BRCA1-associated breast cancers, mammary carcinomas in this new mouse model were ERalpha-negative and of basal epithelial origin. Brca1 deficiency and haploinsufficiency in 60 cM of chromosome 11 caused widespread genome instability as determined by spectral karyotyping analysis. In addition to the verification of the long-range deletion event, the spectral karyotyping analysis revealed that the duplication of the genome and higher degree of aneuploidy occur rather late in tumor progression. Despite chromosomal rearrangements near the Trp53 locus as determined by fluorescence in situ hybridization, the Trp53 gene was transcriptionally active. The analysis of the coding sequence and expression pattern of p53 and p21 suggests that loss-of-heterozygosity of Trp53 caused by somatic mutations contributes to accelerated mammary tumorigenesis in this model.

Characterization of de novo microdeletions involving 17q11.2q12 identified through chromosomal comparative genomic hybridization

High-resolution array-comparative genome hybridization (CGH) is a powerful tool for detection of submicroscopic chromosome deletions and duplications. We describe two patients with mild mental retardation (MR) and de novo microdeletions of 17q11.2q12. Although the deletions did not involve the neurofibromatosis type 1 (NF1) gene, they overlap with long-range deletions of the NF1 region which have been encountered in a small group of NF1 patients with more severe MR. Given the overlap of the deletions in our two patients with the large-sized NF1 microdeletions but not with the more frequent and smaller NF1 deletions, we hypothesize that more than one gene in the 17q11.2q12 region may be involved in MR. We discuss candidate genes for MR within this interval that was precisely defined through array-CGH analysis.

Construction of consecutive deletions of the Escherichia coli chromosome

The minimal set of genetic information necessary and sufficient to sustain a functioning cell contains not only trans-acting genes, but also cis-acting chromosomal regions that cannot be complemented by plasmids carrying these regions. In Escherichia coli (E. coli), only one chromosomal region, the origin of replication has been identified to be cis-acting. We constructed a series of mutants with long-range deletions, and the chromosomal regions containing trans-acting essential genes were deleted in the presence of plasmids complementing the deleted genes. The deleted regions cover all regions of the chromosome except for the origin and terminus of replication. The terminus affects cell growth, but is not essential. Our results indicate that the origin of DNA replication is the only vital, unique cis-acting DNA sequence in the E. coli chromosome necessary for survival.

The molecular genetics of the 22q11-associated schizophrenia

Schizophrenia has a strong genetic component but the mode of inheritance of the disease is complex and in all likelihood involves interaction among multiple genes and also possibly environmental or stochastic factors. A number of studies have shown that the 22q11 deletion syndrome (22q11DS) is a true genetic subtype of schizophrenia and as such may play an extremely important role in deciphering the genetic basis of schizophrenia. Microdeletions of the 22q11 locus are associated with a staggering increased risk to develop schizophrenia. The same locus has also been implicated by some linkage studies. Systematic examination of individual genes from the 1.5 Mb critical region has identified so far the PRODH and ZDHHC8 as strong candidate schizophrenia susceptibility genes from this locus. Discovery of these genes implicates neuromodulatory aminoacids and protein palmitoylation as important for disease development. Other genes, including the gene encoding for COMT, have been implicated by candidate gene approaches. It therefore appears that the 22q11-associated schizophrenia may have the characteristics of a contiguous gene syndrome, where deficiency in more than one gene contributes to the strikingly increased disease risk. Mouse models for individual candidate genes will provide the investigators with the opportunity to start understanding the function of these genes and how they may impact on schizophrenia. Mouse models that carry long-range deletions will likely capture the interactions among the culprit genes and help explain the genetic contribution of this locus to the high risk for schizophrenia. In-depth human and animal model studies of 22q11DS promise to answer critical questions relating to the devastating illness of schizophrenia, whose causes remain largely unknown.