A gap-free genome assembly of Chlamydomonas reinhardtii and detection of translocations induced by CRISPR-mediated mutagenesis

Payne, Zachary L.; Penny, Gervette M; Turner, Tychele N.; Dutcher, Susan K.

doi:10.1016/j.xplc.2022.100493

Cited by 17 publications

(24 citation statements)

References 144 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A pan-genome project has been initiated, targeting genome assemblies for multiple laboratory strains and field isolates. Indeed, concurrent with the publication of this work, chromosome-level assemblies for the field isolates CC-1952 and CC-2931 have been released ( López-Cortegano et al 2022 ), as has the first essentially complete genome for a laboratory strain ( Payne et al 2022 ). As demonstrated herein, many insights can only be gleaned by comparing the genomes of different strains, and we can expect substantial benefits from sequencing additional strains and isolates moving forward.…”

Section: Resultsmentioning

confidence: 99%

The Chlamydomonas Genome Project, version 6: Reference assemblies for mating-type plus and minus strains reveal extensive structural mutation in the laboratory

et al. 2022

View full text Add to dashboard Cite

Five versions of the Chlamydomonas reinhardtii reference genome have been produced over the last two decades. Here we present version 6, bringing significant advances in assembly quality and structural annotations. PacBio-based chromosome-level assemblies for two laboratory strains, CC-503 and CC-4532, provide resources for the plus and minus mating type alleles. We corrected major misassemblies in previous versions and validated our assemblies via linkage analyses. Contiguity increased over ten-fold and >80% of filled gaps are within genes. We used Iso-Seq and deep RNA-seq datasets to improve structural annotations, and updated gene symbols and textual annotation of functionally characterized genes via extensive manual curation. We discovered that the cell wall-less classical reference strain CC-503 exhibits genomic instability potentially caused by deletion of the helicase RECQ3, with major structural mutations identified that affect >100 genes. We therefore present the CC-4532 assembly as the primary reference, although this strain also carries unique structural mutations and is experiencing rapid proliferation of a Gypsy retrotransposon. We expect all laboratory strains to harbor gene-disrupting mutations, which should be considered when interpreting and comparing experimental results. Collectively, the resources presented here herald a new era of Chlamydomonas genomics and will provide the foundation for continued research in this important reference organism.

show abstract

Section: Resultsmentioning

confidence: 99%

The Chlamydomonas Genome Project, version 6: Reference assemblies for mating-type plus and minus strains reveal extensive structural mutation in the laboratory

et al. 2022

View full text Add to dashboard Cite

show abstract

“…We obtained three independent strains from two separate transformations. These three strains were renamed pf23-2, pf23-3, and pf23-4 [107] (Supplemental Fig S5). Immunofluorescence analysis of gametic wild-type and pf23 mutant strains show that the pf23-1 strain assembles cilia that are 2.14 +/- 0.86 microns long, while the pf23 null strains assemble cilia that average 0.87 +/- 0.23 microns in length (Fig 8A).…”

Section: Resultsmentioning

confidence: 99%

“…Meiotic analysis of all three strains showed that pf23-2 and pf23-3 produced meiotic inviability, while crosses with pf23-4 produced primarily tetrads with four viable progeny. Long-range sequencing of pf23-2 and pf23-3 showed that these strains contain translocations between the PF23 gene and chromosome 5 and 3, respectively [107]. Because pf23-4 did not show any meiotic abnormalities, it is unlikely that it possesses a translocation.…”

Section: Resultsmentioning

confidence: 99%

“…The two colonies were then digested with restriction enzymes Nde I and I- Sce I to confirm the presence of the PLM099 backbone and the PF13 gene (Supplemental Fig 2C). DNA from PF13:: YFP-FLAG #2 was isolated from a 4 ml overnight culture was used to transform pf13-1 as described [107]. PCR to detect the presence of the transgene was performed on a subset of colonies (Supplemental Fig 2D) that showed a rescued phenotype of swimming in liquid 96-well plates under a dissecting microscope ( pf13-1 is non-motile.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Reduced dosage of multiple dynein arm preassembly factors limits cilia regeneration inChlamydomonas reinhardtii

Penny,

Dutcher

2023

Preprint

Self Cite

View full text Add to dashboard Cite

Motile cilia are complex organelles comprised of >800 structural proteins. Variants in 58 of these genes cause primary ciliary dyskinesia (PCD) in humans. We used a second-site non-complementation approach in diploidChlamydomonas reinhardtiistrains to investigate whether reduced dosage of different combinations of motile cilia proteins affects cilia growth / regeneration. Temperature-sensitive mutants in intraflagellar transport genes fail to complement in heterozygous diploids at the restrictive temperature, which may be due to poisonous interactions or a reduction in protein levels. Diploid strains heterozygous for 211 double mutant combinations in 21 genes are phenotypically recessive when unperturbed and do not show SSNC. Consequently, we developed a sensitized screen. When protein synthesis is inhibited, cells utilize the existing cytoplasmic pool of proteins, which is insufficient to build full-length cilia. Six double heterozygous strains in dynein arm preassembly factors (DNAAFs)pf13, pf23, wdr92,andoda8regrow cilia that are shorter than wild-type diploids, which suggests that double heterozygosity limits the pool of assembled dynein arms further. We isolated a new null allele (pf23-4)that shows a more severe loss of ODAs and IDAs in cilia than the previously studiedpf23-1hypomorphic allele. We also identified a new role for PF23 in cytoplasmic modification of IC138, protein of the I1/finner dynein arm. In our SSNC assay, thepf23-4allele also exhibits a more severe phenotype thanpf23-1. Whole cell extract immunoblots show a reduction of PF23 protein to one-half of wild-type levels in both single and double heterozygous genotypes. Thepf13mutant shows SSNC with mutants in two outer dynein arm structural proteins, ODA6, andODA9. We suggest that reduction of multiple dynein preassembly factors limits the pool of assembled dynein arms needed for cilia assembly and regeneration. Our data support PF23 as a scaffolding hub protein involved in dynein arm assembly.Author SummaryMotile cilia are essential for movement of cells and fluids. In humans, motile cilia defects cause primary ciliary dyskinesia (PCD), a rare disease characterized by recurring respiratory infections, left-right asymmetry defects, ear infections, and infertility. Many genes are involved in motile cilia function and assembly. We useChlamydomonas reinhardtiito study the effects of changes in protein levels in different combinations of proteins needed for motile cilia. Our results show that the proteins that help fold and assemble the dynein motors in the cytoplasm are sensitive to changes in the dosage of these preassembly factors. Reductions in proteins involved in multiple steps of the pathway prevents efficient dynein preassembly and cilia regeneration under stressful cellular conditions.

show abstract

“…This sequence information had been used for comparative genomics, defining genes potentially associated with photosynthesis (Grossman et al, 2019; Karpowicz et al, 2011) and cilia and basal bodies (Li et al, 2004; Stolc et al, 2005). The genomes of other Chlamydomonas reinhardtii strains have been sequenced and assembled, defining 17 centromeres, rDNA arrays, and over 50 fragments of organellar DNA inserted into the nuclear genome (Payne et al, 2023), with new assemblies validating gene location with genetic linkage analyses (Craig et al, 2023). These studies increased the contiguity of the genome, filling in many gaps, while Iso‐Seq, high coverage RNA‐seq, and manual curation have improved gene models and annotation.…”

Section: The Genomementioning

confidence: 99%

Chlamydomonas: Fast tracking from genomics

Findinier

Grossman

2023

Journal of Phycology

View full text Add to dashboard Cite

Elucidating biological processes has relied on the establishment of model organisms, many of which offer advantageous features such as rapid axenic growth, extensive knowledge of their physiological features and gene content, and the ease with which they can be genetically manipulated. The unicellular green alga Chlamydomonas reinhardtii has been an exemplary model that has enabled many scientific breakthroughs over the decades, especially in the fields of photosynthesis, cilia function and biogenesis, and the acclimation of photosynthetic organisms to their environment. Here, we discuss recent molecular/technological advances that have been applied to C. reinhardtii and how they have further fostered its development as a “flagship” algal system. We also explore the future promise of this alga in leveraging advances in the fields of genomics, proteomics, imaging, and synthetic biology for addressing critical future biological issues.

show abstract

A gap-free genome assembly of Chlamydomonas reinhardtii and detection of translocations induced by CRISPR-mediated mutagenesis

Cited by 17 publications

References 144 publications

The Chlamydomonas Genome Project, version 6: Reference assemblies for mating-type plus and minus strains reveal extensive structural mutation in the laboratory

The Chlamydomonas Genome Project, version 6: Reference assemblies for mating-type plus and minus strains reveal extensive structural mutation in the laboratory

Reduced dosage of multiple dynein arm preassembly factors limits cilia regeneration inChlamydomonas reinhardtii

Chlamydomonas: Fast tracking from genomics

Contact Info

Product

Resources

About