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Chromosomal rearrangements can initiate and drive cancer progression, yet it has been challenging to evaluate their impact, especially in genetically heterogeneous solid cancers. To address this problem we developed HiDENSEC, a new computational framework for analyzing chromatin conformation capture in heterogeneous samples, which can infer somatic copy number alterations, characterize large-scale chromosomal rearrangements, and estimate cancer cell fractions. We validated HiDENSEC within silicoandin vitrocontrols, and then characterized chromosome-scale evolution during melanoma progression in formalin-fixed tumor samples from three patients. The resulting comprehensive annotation of the genomic events includes copy number neutral translocations that disrupt tumor suppressor genes such as NF1, whole chromosome arm exchanges that result in loss of CDKN2A, and whole-arm copy-number neutral loss of homozygosity involving PTEN. These findings show that large-scale chromosomal rearrangements occur throughout cancer evolution and characterizing these events yields insights into drivers of melanoma progression.

This protocol describes the procedure or preparing MEF-cultured human pluripotent stem cells (hPSCs) for the delivery of plasmids, mRNA or ribonucleoprotein (RNP) using nucleofection. General notes 1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs. 2. This protocol is to prepare cells for protocol nucleofection of hPSCs. Before starting, familiarize yourself with the protocol and the required preparations. A detailed protocol on maintaining MEF-cultured hPSCs can be found in the collection "Maintenance and inactivation of mouse embryonic fibroblasts (MEFs) as feeder cells for human pluripotent stem cell culture;" doi: 3. Detailed protocols for preparing plasmids, RNA, and RNP for nucleofection can be found in the collection "Nucleofection (Amaxa) and electroporation (Biorad) of hPSCs." A link to this collection can be found in the title section of this protocol, located above

This collection describes the standard procedure for the delivery of plasmids, mRNA or ribonucleoprotein (RNP) into human pluripotent stem cells (hPSCs) using nucleofection or electroporation. Collection Overview Preparing MEF-cultured hPSCs for nucleofection Preparing Feeder-free hPSCs for nucleofection Preparing plasmids for nucleofection Preparing mRNA for nucleofection In vitro assembling of RNP for nucleofection Nucleofection of hPSCs Electroporation of hPSCs A. Preparation of MEFs-cultured hPSCs for electroporation B. Preparing plasmids for electroporation C. Electroporation FACS-based enrichment of transfected cells A. Preparation of samples for FACS sorting B. After FACS General notes Throughout these protocols, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.

The recent development of prime editing (PE) genome engineering technologies has the potential to significantly simplify the generation of human pluripotent stem cell (hPSC)-based disease models. PE is a multicomponent editing system that uses a Cas9-nickase fused to a reverse transcriptase (nCas9-RT) and an extended PE guide RNA (pegRNA). Once reverse transcribed, the pegRNA extension functions as a repair template to introduce precise designer mutations at the target site. Here, we systematically compared the editing efficiencies of PE to conventional gene editing methods in hPSCs. This analysis revealed that PE is overall more efficient and precise than homology-directed repair of site-specific nuclease-induced double-strand breaks. Specifically, PE is more effective in generating heterozygous editing events to create autosomal dominant disease-associated mutations. By stably integrating the nCas9-RT into hPSCs we achieved editing efficiencies equal to those reported for cancer cells, suggesting that the expression of the PE components, rather than cell-intrinsic features, limit PE in hPSCs. To improve the efficiency of PE in hPSCs, we optimized the delivery modalities for the PE components. Delivery of the nCas9-RT as mRNA combined with synthetically generated, chemically-modified pegRNAs and nicking guide RNAs improved editing efficiencies up to 13-fold compared with transfecting the PE components as plasmids or ribonucleoprotein particles. Finally, we demonstrated that this mRNA-based delivery approach can be used repeatedly to yield editing efficiencies exceeding 60% and to correct or introduce familial mutations causing Parkinson's disease in hPSCs.From muscles to nerves, our body is formed of many kinds of cells which can each respond slightly differently to the same harmful genetic changes. Understanding the exact relationship between mutations and cell-type specific function is essential to better grasp how conditions such as Parkinson’s disease or amyotrophic lateral sclerosis progress and can be treated. Stem cells could be an important tool in that effort, as they can be directed to mature into many cell types in the laboratory. Yet it remains difficult to precisely introduce disease-relevant mutations in these cells. To remove this obstacle, Li et al. focused on prime editing, a cutting-edge ‘search and replace’ approach which can introduce new genetic information into a specific DNA sequence. However, it was unclear whether this technique could be used to efficiently create stem cell models of human diseases. A first set of experiments showed that prime editing is superior to conventional approaches when generating mutated genes in stem cells. Li et al. then further improved the efficiency and precision of the method by tweaking how prime editing components are delivered into the cells. The refined approach could be harnessed to quickly generate large numbers of stem cells carrying mutations associated with Parkinson’s disease; crucially, prime editing could then also be used to revert a mutated gene back to its healthy form. The improved prime editing approach developed by Li et al. removes a major hurdle for scientists hoping to use stem cells to study genetic diseases. This could potentially help to unlock progress in how we understand and ultimately treat these conditions.

This protocol describes a standard procedure culturing and transfecting HEK293T cells Protocol overview: A. Culturing HEK293T cells B. Transfection of HEK293T cells with Lipofectamine 2000

This protocol describes the experimental procedure used to measure single cell survival rates post nucleofection of human pluripotent stem cells (hPSCs). General Notes: 1. Throughout these protocols, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.

This protocol describes the standard procedure preparing cell pellets from human pluripotent stem cells (hPSCs) cultured on MEFs for outsourced aCGH karyotyping. General Notes: 1. Throughout these protocols, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.

Telomerase regulation and telomere shortening act as a strong tumor suppressor mechanism in human somatic cells. Point mutations in the promoter of telomerase reverse transcriptase (TERT) are the most frequent non-coding mutation in cancer. These TERT promoter mutations (TPMs) create de novo ETS factor binding sites upstream of the start codon of the gene, which can be bound by different ETS factors. TPMs can occur early during tumorigenesis and are thought to be among the first mutations in melanoma, glioblastoma and hepatocellular carcinoma. Despite their association with increased TERT levels, TPMs do not prohibit telomere shortening and TPM-harboring cancers present with short telomeres. Their short telomere length combined with their high prevalence and specificity for cancer makes TPMs an attractive target for future therapeutic exploitation of telomerase inhibition and telomere deprotection-induced cell death.

This protocol describes the standard procedure for staining pluripotency markers, e.g. OCT4, SSEA4, alkaline phosphatase, and etc. on human pluripotent stem cells (hPSCs). Protocol overview A. Immunofluorescence staining B. Alkaline phosphatase staining General notes Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.

This protocol describes the standard procedure preparing feeder-free human pluripotent stem cells (hPSCs) for nucleofection. General notes 1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs. 2. This protocol is to prepare cells for protocol nucleofection of hPSCs. Before starting, familiarize yourself with the protocol and the required preparations. A detailed protocol on maintaining MEF-cultured hPSCs can be found in the collection "Maintenance and inactivation of mouse embryonic fibroblasts (MEFs) as feeder cells for human pluripotent stem cell culture;" doi: 3. Detailed protocols for preparing plasmids, RNA, and RNP for nucleofection can be found in the collection "Nucleofection (Amaxa) and electroporation (Biorad) of hPSCs." A link to this collection can be found in the title section of this protocol, located above.

This collection describes the standard procedure for the delivery of plasmids, mRNA or ribonucleoprotein (RNP) into human pluripotent stem cells (hPSCs) using nucleofection or electroporation. Collection Overview Preparing MEF-cultured hPSCs for nucleofection Preparing Feeder-free hPSCs for nucleofection Preparing plasmids for nucleofection Preparing mRNA for nucleofection In vitro assembling of RNP for nucleofection Nucleofection of hPSCs Electroporation of hPSCs A. Preparation of MEFs-cultured hPSCs for electroporation B. Preparing plasmids for electroporation C. Electroporation FACS-based enrichment of transfected cells A. Preparation of samples for FACS sorting B. After FACS General notes Throughout these protocols, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.

This protocol describes a standard procedure for subcloning of genotype-confirmed human pluripotent stem cells (hPSCs). General notes: 1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.

This protocol describes the standard procedure of thawing human pluripotent stem cells (hPSCs) on inactivated mouse embryonic fibroblasts (MEFs). General notes 1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs. 2. Until otherwise indicated, hPSCs are routinely grown in a humidified cell culture incubator under “low” oxygen conditions. We have successfully maintained hPSCs using either 3% O2 (3% O2, 5% CO2) or 5% O2 (5% O2, 5% CO2) conditions. 3. While freezing hPSCs as single cell solution (using Rock Inhibitor) results in better cell recovery, some laboratories prefer freezing of hPSCs as cell clusters. We have used both approaches and do not observe obvious differences.

This protocol describes the procedure FACS-based enrichment of transfected human pluripotent stem cells (hPSCs). Protocol overview A. Preparation of samples for FACS sorting B. After FACS General notes 1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs. 2. Dependent on the genome editing strategy, we use either drug selection or FACS sorting to enrich transfected cell populations based on the presence of a fluorescent protein (either as part of the transfected plasmids or through co-transfection of a fluorescent protein expressing plasmid). 3. Either drug selection or FACS sorting will be usually performed 48 – 72 hours after electroporation/nucleofection. 4. Carefully plan the timeline of FACS experiments and schedule required FACS sorting time at core facility (requires approximately 45 min sorting time per sample) 5. Every FACS sorting experiment requires an additional non-electroporated well of hPSCs (parental cell line) as a negative control to determine the appropriate FACS gates.

This protocol describes the procedure for the in vitro assembly of ribonucleoprotein (RNP) which can be delivered into human pluripotent stem cells (hPSCs) using nucleofection. General notes 1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.

This protocol describes the thawing of mouse embryonic fibroblasts (MEFs) as feeder cells for human pluripotent stem cell (hPSC) culture. General notes 1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs. 2. MEFs were obtained as described in Manipulating the Mouse Embryo: A Laboratory Manual, Third Edition (ISBN: 0879695919)

This collection describes a standard procedure for isolating single human pluripotent stem cell (hPSC) clones in a high-throughput way. This collection follows nucleofection of hPSCs as described in detail in the collection "Nucleofection (Amaxa) andelectroporation(Biorad) of hPSCs;" dx.doi.org/10.17504/protocols.io.b4qnqvve Collection overview Seeding nucleofected hPSCs in 96-well plates using limited dilution Duplicating 96-well plate-cultured hPSCs clones Subcloning of genotype-confirmed hPSCs clones General notes 1. Throughout these protocols, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.

This protocol describes the standard procedure for the preparation of plasmids to be delivered into human pluripotent stem cells (hPSCs) using nucleofection. General notes 1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.

This protocol describes the process of harvesting and irradiating mouse embryonic fibroblasts (MEFs) to use as feeder cells for human pluripotent stem cell (hPSC) culture. General notes 1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs. 2. MEFs were obtained as described in Manipulating the Mouse Embryo: A Laboratory Manual, Third Edition (ISBN: 0879695919)

This protocol describes the freezing of mouse embryonic fibroblasts (MEFs), which can later be used as feeder cells for human pluripotent stem cell (hPSC) culture. General notes 1. This protocol can be used to freeze MEFs before or after inactivation. 2. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs. 3. MEFs were obtained as described in Manipulating the Mouse Embryo: A Laboratory Manual, Third Edition (ISBN: 0879695919)

This protocol describes a standard procedure for seeding nucleofected human pluripotent stem cells in 96-well plates using limited dilution. This protocol follows nucleofection of hPSCs as described in detail in the collection "Nucleofection (Amaxa) andelectroporation(Biorad) of hPSCs;" dx.doi.org/10.17504/protocols.io.b4qnqvve General notes 1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.

This protocol describes the process of using Mitomycin C to inactivate mouse embryonic fibroblasts (MEFs), which can then be used as feeder cells for human pluripotent stem cell (hPSC) culture. General notes 1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs. 2. MEFs were obtained as described in Manipulating the Mouse Embryo: A Laboratory Manual, Third Edition (ISBN: 0879695919)

This protocol describes a procedure for amplifying targeted genomic regions using a PCR reaction

This protocol describes the procedure or preparing MEF-cultured human pluripotent stem cells (hPSCs) for the delivery of plasmids, mRNA or ribonucleoprotein (RNP) using nucleofection. General notes 1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs. 2. This protocol is to prepare cells for protocol nucleofection of hPSCs. Before starting, familiarize yourself with the protocol and the required preparations. A detailed protocol on maintaining MEF-cultured hPSCs can be found in the collection "Maintenance and inactivation of mouse embryonic fibroblasts (MEFs) as feeder cells for human pluripotent stem cell culture;" doi: 3. Detailed protocols for preparing plasmids, RNA, and RNP for nucleofection can be found in the collection "Nucleofection (Amaxa) and electroporation (Biorad) of hPSCs." A link to this collection can be found in the title section of this protocol, located above

This collection contains protocols which describe the standard procedure of culturing human pluripotent stem cells (hPSCs) on inactivated mouse embryonic fibroblasts (MEFs). Collection overview Thawing of hPSCs grown on MEFs Passaging of hPSCs grown on MEFs Freezing of hPSCs grown on MEFs A. Freezing of hPSCs as single cell suspension using trypsin B. Freezing of hPSCs as cell aggregates using collagenase General notes 1. Throughout these protocols, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs. 2. Until otherwise indicated, hPSCs are routinely grown in a humidified cell culture incubator under “low” oxygen conditions. We have successfully maintained hPSCs using either 3% O2 (3% O2, 5% CO2) or 5% O2 (5% O2, 5% CO2) conditions. 3. While freezing hPSCs as single cell solution (using Rock Inhibitor) results in better cell recovery, some laboratories prefer freezing of hPSCs as cell clusters. We have used both approaches and do not observe obvious differences. 4. While bulk/collagenase passaging is used for routine maintenance of hPSC cultures, manual/microdissection passaging is used to enrich for undifferentiated hPSC colonies (“clean-up” of culture based on undifferentiated hPSC colony morphology) or to expand (“pick”) individual colonies (e.g.,for clonal expansion of individual targeted cells in the process of establishing genome edited cell lines). Manual passaging/ microdissection requires (i) the identification and discrimination of undifferentiated and differentiated hPSC colonies and (ii) the capacity to excise the undifferentiated cells and transfer them to a new plate and can be performed using various approaches as established in many hPSC laboratories.

This protocol describes the standard procedure for preparing mRNA to be delivered into human pluripotent stem cells (hPSCs) using nucleofection. General notes 1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.

This protocol describes a standard procedure for duplicating 96-well plate-cultured human pluripotent stem cells (hPSCs). General notes: 1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs

This protocol describes the process of using CRISPResso to analyze Next Generation Sequencing (NGS) data from CRISPR-targeted, genome-edited samples.

This protocol describes the standard procedure of using collagenase to passage human pluripotenct stem cells (hPSCs) on inactivated mouse embryonic fibroblasts (MEFs). General notes 1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs. 2. Until otherwise indicated, hPSCs are routinely grown in a humidified cell culture incubator under “low” oxygen conditions. We have successfully maintained hPSCs using either 3% O2 (3% O2, 5% CO2) or 5% O2 (5% O2, 5% CO2) conditions. 3. While bulk/collagenase passaging is used for routine maintenance of hPSC cultures, manual/microdissection passaging is used to enrich for undifferentiated hPSC colonies (“clean-up” of culture based on undifferentiated hPSC colony morphology) or to expand (“pick”) individual colonies (e.g.,for clonal expansion of individual targeted cells in the process of establishing genome edited cell lines). Manual passaging/ microdissection requires (i) the identification and discrimination of undifferentiated and differentiated hPSC colonies and (ii) the capacity to excise the undifferentiated cells and transfer them to a new plate and can be performed using various approaches as established in many hPSC laboratories.

This protocol describes a standard procedure for preparing crude cell lysate which can be further analyzed by PCR. Protocol overview: A. Preparing crude cell lysate directly from hPSCs culture B. Preparing crude cell lysate from dissociated cells C. Preparing cell lysate from FACS-sorted cells (small cell number) General notes 1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.

This protocol describes the standard procedure for the delivery of plasmids into human pluripotent stem cells (hPSCs) using electroporation. Protocol Overview A. Preparation of MEFs-cultured hPSCs for electroporation B. Preparing plasmids for electroporation C. Electroporation General notes 1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.

This protocol describes the standard procedure for the delivery of plasmids, mRNA or ribonucleoprotein (RNP) into human pluripotent stem cells (hPSCs) using nucleofection. General notes Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.

This collection describes a standard genotyping procedure using next generation sequencing (NGS) in the Hockemeyer lab. Collection overview Preparing of genomic DNA from in vitro cultured cells A. Preparing crude cell lysate directly from hPSCs culture B. Preparing crude cell lysate from dissociated cells C. Preparing cell lysate from FACS-sorted cells Amplifying the target genomic region by PCR Preparing samples for NGS CRISPResso analysis General notes: 1. Throughout these protocols, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.

This protocol describes the expansion of mouse embryonic fibroblasts (MEFs) as feeder cells for human pluripotent stem cell (hPSC) culture. General notes 1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs. 2. MEFs were obtained as described in Manipulating the Mouse Embryo: A Laboratory Manual, Third Edition (ISBN: 0879695919)

This protocol describes the standard procedure of freezing human pluripotent stem cells (hPSCs), which were grown on inactivated mouse embryonic fibroblasts (MEFs). General notes 1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs. 2. Until otherwise indicated, hPSCs are routinely grown in a humidified cell culture incubator under “low” oxygen conditions. We have successfully maintained hPSCs using either 3% O2 (3% O2, 5% CO2) or 5% O2 (5% O2, 5% CO2) conditions. 3. While freezing hPSCs as single cell solution (using Rock Inhibitor) results in better cell recovery, some laboratories prefer freezing of hPSCs as cell clusters. We have used both approaches and do not observe obvious differences.

This protocol describes the preparation of mouse embryonic fibroblasts (MEFs) as feeder cells for human pluripotent stem cell (hPSC) culture. Protocol overview A. Starting with frozen irradiated or Mitomycin C inactivated MEFs (optional) B. Starting with fresh irradiated or Mitomycin C inactivated MEFs General notes 1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs. 2. Either fresh (start at step 4) or frozen stocks of irradiated or Mitomycin C inactivated MEFs can be used to prepare hPSC feeder cells. 3. The indicated MEF density are recommended starting densities and might have to be adjusted for each hPSC line and hPSC medium formulation (KSR, serum-free versus serum-containing medium). 4. MEFs were obtained as described in Manipulating the Mouse Embryo: A Laboratory Manual, Third Edition (ISBN: 0879695919)

This protocol describes the procedure of adapting human pluripotent stem cells (hPSCs) to feeder-free culturing conditions using mTeSR-plus or StemFlex General Notes Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs. Until otherwise indicated, feeder-free hPSCs are routinely grown in a humidified cell culture incubator under “low” oxygen conditions. We have successfully maintained hPSCs using either 3% O2 (3% O2, 5% CO2) or 5% O2 (5% O2, 5% CO2) conditions. We have routinely maintained feeder-free cells in either mTeSR-plus or StemFlex. However, these two mediums are not interchangeable. Pick one and stick to it. We have routinely maintained feeder-free hPSC cultures on VTN, Matrigel and Geltrex--coated cell culture plates without observing obvious differences.

This protocol describes the process of freezing feeder-free human pluripotent stem cells (hPSCs) using Accutase or ReLeSR Protocol overview: A. Accutase B. ReLeSR General Notes: For this protocol, hPSCs refers collectively to hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs. Until otherwise indicated, feeder-free hPSCs are routinely grown in a humidified cell culture incubator under “low” oxygen conditions. We have successfully maintained hPSCs using either 3% O2 (3% O2, 5% CO2) or 5% O2 (5% O2, 5% CO2) conditions. We have routinely maintained feeder-free cells in either mTeSR-plus or StemFlex, however these two mediums are not interchangeable. Pick one and stick to it. We have routinely maintained feeder-free hPSC cultures on VTN, Matrigel and Geltrex coated cell culture plates without observing obvious differences.

This collection describes the standard procedure of feeder-free culturing of human pluripotent stem cells (hPSCs) using mTeSR-plus or StemFlex Collection overview Coating plates A. VTN B. Matrigel C. Geltrex Adapting hPSCs cultured on MEFs to feeder-free system Thawing of feeder-free hPSCs Passaging of feeder-free hPSCs A. Accutase B. ReLeSR Freezing of feeder-free hPSCs A. Accutase B. ReLeSR General Notes Throughout these protocols, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs. Until otherwise indicated, feeder-free hPSCs are routinely grown in a humidified cell culture incubator under “low” oxygen conditions. We have successfully maintained hPSCs using either 3% O2 (3% O2, 5% CO2) or 5% O2 (5% O2, 5% CO2) conditions. We have routinely maintained feeder-free cells in either mTeSR-plus or StemFlex. However, these two media are not interchangeable. Pick one and stick to it. We have routinely maintained feeder-free hPSC cultures on VTN, Matrigel and Geltrex-coated cell culture plates without observing obvious differences. We have routinely passaged feeder-free hPSCs using either Accutase (as single cell suspension) or ReLeSR (as cell aggregates) without observing obvious differences.

This protocol describes the procedure of thawing feeder-free human pluripotent stem cells (hPSCs) using mTeSR-plus or StemFlex General Notes Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs. Until otherwise indicated, feeder-free hPSCs are routinely grown in a humidified cell culture incubator under “low” oxygen conditions. We have successfully maintained hPSCs using either 3% O2 (3% O2, 5% CO2) or 5% O2 (5% O2, 5% CO2) conditions. We have routinely maintained feeder-free cells in either mTeSR-plus or StemFlex. However, these two mediums are not interchangeable. Pick one and stick to it. We have routinely maintained feeder-free hPSC cultures on VTN, Matrigel and Geltrex--coated cell culture plates without observing obvious differences.

This protocol describes the process of coating plates using either VTN, Matrigel or Geltrex for use in culturing of feeder-free human pluripotent stem cells (hPSCs) Protocol overview Coating plates A. VTN B. Matrigel C. Geltrex General Notes: Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.

This protocol describes the standard procedure to knock-in constructs to the AAVS1 safe harbor locus in hPSCs. General Notes: 1. The AAVS1 knock-in construct, AAVS1-SA-neo-CAGGS-PE2-2A-GFP, can be found at AddGene (Catalog: 180014, RRID:Addgene_180014) 2. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.

Frogs are an ecologically diverse and phylogenetically ancient group of living amphibians that include important vertebrate cell and developmental model systems, notably the genus Xenopus. Here we report a high-quality reference genome sequence for the western clawed frog, Xenopus tropicalis, along with draft chromosome-scale sequences of three distantly related emerging model frog species, Eleutherodactylus coqui, Engystomops pustulosus and Hymenochirus boettgeri. Frog chromosomes have remained remarkably stable since the Mesozoic Era, with limited Robertsonian (i.e., centric) translocations and end-to-end fusions found among the smaller chromosomes. Conservation of synteny includes conservation of centromere locations, marked by centromeric tandem repeats associated with Cenp-a binding, surrounded by pericentromeric LINE/L1 elements. We explored chromosome structure across frogs, using a dense meiotic linkage map for X. tropicalis and chromatin conformation capture (HiC) data for all species. Abundant satellite repeats occupy the unusually long (∼20 megabase) terminal regions of each chromosome that coincide with high rates of recombination. Both embryonic and differentiated cells show reproducible association of centromeric chromatin, and of telomeres, reflecting a Rabl configuration similar to the “bouquet” structure of meiotic cells. Our comparative analyses reveal 13 conserved ancestral anuran chromosomes from which contemporary frog genomes were constructed.

Mutations in the TINF2 gene, encoding the shelterin protein TIN2, cause telomere shortening and the inherited bone marrow (BM) failure syndrome dyskeratosis congenita (DC). A lack of suitable model systems limits the mechanistic understanding of telomere shortening in the stem cells and thus hinders the development of treatment options for BM failure. Here, we endogenously introduced TIN2-DC mutations in human embryonic stem cells (hESCs) and human hematopoietic stem and progenitor cells (HSPCs) to dissect the disease mechanism and identify a gene-editing strategy that rescued the disease phenotypes. The hESCs with the T284R disease mutation exhibited the short telomere phenotype observed in DC patients. Yet, telomeres in mutant hESCs did not trigger DNA damage responses at telomeres or show exacerbated telomere shortening when differentiated into telomerase-negative cells. Disruption of the mutant TINF2 allele by introducing a frameshift mutation in exon 2 restored telomere length in stem cells and the replicative potential of differentiated cells. Similarly, we introduced TIN2-DC disease variants in human HSPCs to assess the changes in telomere length and proliferative capacity. Lastly, we showed that editing at exon 2 of TINF2 that restored telomere length in hESCs could be generated in TINF2-DC patient HSPCs. Our study demonstrates a simple genetic intervention that rescues the TIN2-DC disease phenotype in stem cells and provides a versatile platform to assess the efficacy of potential therapeutic approaches in vivo.

SummaryRegular surveillance testing of asymptomatic individuals for SARS-CoV-2 has played a vital role in SARS-CoV-2 outbreak prevention on college and university campuses. Here we describe the voluntary saliva testing program instituted at the University of California, Berkeley during an early period of the SARS-CoV-2 pandemic in 2020. The program was administered as a research study ahead of clinical implementation, enabling us to launch surveillance testing while continuing to optimize the assay. Results of both the testing protocol itself and the study participants’ experience show how the program succeeded in providing routine, robust testing capable of contributing to outbreak prevention within a campus community and offer strategies for encouraging participation and a sense of civic responsibility.

Telomere maintenance is essential for the long-term proliferation of human pluripotent stem cells, while their telomere length set point determines the proliferative capacity of their differentiated progeny. The shelterin protein TPP1 is required for telomere stability and elongation, but its role in establishing a telomere length set point remains elusive. Here, we characterize the contribution of the shorter isoform of TPP1 (TPP1S) and the amino acid L104 outside the TEL patch, TPP1's telomerase interaction domain, to telomere length control. We demonstrate that cells deficient for TPP1S (TPP1S knockout [KO]), as well as the complete TPP1 KO cell lines, undergo telomere shortening. However, TPP1S KO cells are able to stabilize short telomeres, while TPP1 KO cells die. We compare these phenotypes with those of TPP1L104A/L104A mutant cells, which have short and stable telomeres similar to the TPP1S KO. In contrast to TPP1S KO cells, TPP1L104A/L104A cells respond to increased telomerase levels and maintain protected telomeres. However, TPP1L104A/L104A shows altered sensitivity to expression changes of shelterin proteins suggesting the mutation causes a defect in telomere length feedback regulation. Together this highlights TPP1L104A/L104A as the first shelterin mutant engineered at the endogenous locus of human stem cells with an altered telomere length set point.

Closely related muntjac deer show striking karyotype differences. Here we describe chromosome-scale genome assemblies for Chinese and Indian muntjacs, Muntiacus reevesi (2n = 46) and Muntiacus muntjak vaginalis (2n = 6/7), and analyze their evolution and architecture. The genomes show extensive collinearity with each other and with other deer and cattle. We identified numerous fusion events unique to and shared by muntjacs relative to the cervid ancestor, confirming many cytogenetic observations with genome sequence. One of these M. muntjak fusions reversed an earlier fission in the cervid lineage. Comparative Hi-C analysis showed that the chromosome fusions on the M. muntjak lineage altered long-range, three-dimensional chromosome organization relative to M. reevesi in interphase nuclei including A/B compartment structure. This reshaping of multi-megabase contacts occurred without notable change in local chromatin compaction, even near fusion sites. A few genes involved in chromosome maintenance show evidence for rapid evolution, possibly associated with the dramatic changes in karyotype., Mudd et al. provide chromosome-scale genome assemblies for the Chinese and Indian muntjac deer and demonstrate evidence for rapid evolution of genes involved in chromosome maintenance. Their comparative analyses generate new insights into rapid karyotype evolution.

Dopamine neurons project to diverse regions throughout the brain to modulate various brain processes and behaviors. It is increasingly appreciated that dopamine neurons are heterogeneous in their gene expression, circuitry, physiology, and function. Current approaches to target dopamine neurons are largely based on single gene drivers, which either label all dopamine neurons, or mark a sub-set but concurrently label non-dopaminergic neurons. Here we establish a novel mouse line in which Flp recombinase is knocked-in to the endogenousSlc6a3(dopamine active transporter, DAT) locus. DAT-Flp mice can be used with various Cre-expressing mouse lines to efficiently and selectively label dopaminergic subpopulations using Cre/Flp-dependent intersectional strategies. We demonstrate the utility of this approach by crossing DAT-Flp mice with NEX-Cre mice, to specifically labelNeurod6-expressing dopamine neurons that project to the nucleus accumbens medial shell. DAT-Flp mice represent a novel tool, which will help parse the diverse functions mediated by dopaminergic circuits.