Surrogate Reporters Research Articles

Enrichment of prime-edited mammalian cells with surrogate PuroR reporters

Prime editing is a programmable genetic method that can precisely generate any desired small-scale variations in cells without requiring double-strand breaks and DNA donors. However, higher editing efficiency is greatly desirable for wide practical applications. In this study, we developed a target-specific prime editing reporter (tsPER) and a universal prime editing reporter (UPER) to facilitate rapid selection of desired edited cells through puromycin screening. The modification efficiency of HEK3_i1CTT_d5G in HEK293T cells improved from 36.37 % to 64.84 % with the incorporation of tsPER. The target sequence of interested genes could be custom inserted into a selection cassette in tsPER to establish personalized reporters. The UPER demonstrated PE3 editing efficiency up to 74.49 % on HEK3_i1CTT_d5G and 73.52 % on HEK3_i1His6, achieved through co-selection with an additional pegRNA (puro) to repair the mutant PuroR cassette. Overall, tsPER and UPER robustly improved the efficiency of prime editing. Both of these approaches expand enrichment strategies for genomically modified cells and accelerate the generation of genetically modified models.

Efficient CRISPR/Cas9-mediated gene editing in mammalian cells by the novel selectable traffic light reporters

CRISPR/Cas9 is a powerful tool for gene editing in various cell types and organisms. However, it is still challenging to screen genetically modified cells from an excess of unmodified cells. Our previous studies demonstrated that surrogate reporters can be used for efficient screening of genetically modified cells. Here, we developed two novel traffic light screening reporters, puromycin-mCherry-EGFP (PMG) based on single-strand annealing (SSA) and homology-directed repair (HDR), respectively, to measure the nuclease cleavage activity within transfected cells and to select genetically modified cells. We found that the two reporters could be self-repaired coupling the genome editing events driven by different CRISPR/Cas nucleases, resulting in a functional puromycin-resistance and EGFP selection cassette that can be afforded to screen genetically modified cells by puromycin selection or FACS enrichment. We further compared the novel reporters with different traditional reporters at several endogenous loci in different cell lines, for the enrichment efficiencies of genetically modified cells. The results indicated that the SSA-PMG reporter exhibited improvements in enriching gene knockout cells, while the HDR-PMG system was very useful in enriching knock-in cells. These results provide robust and efficient surrogate reporters for the enrichment of CRISPR/Cas9-mediated editing in mammalian cells, thereby advancing basic and applied research.

Dual Cre and Dre recombinases mediate synchronized lineage tracing and cell subset ablation in vivo

Genetic technology using site-specific recombinases, such as the Cre-loxP system, has been widely employed for labeling specific cell populations and for studying their functions in vivo. To enhance the precision of cell lineage tracing and functional study, a similar site-specific recombinase system termed Dre-rox has been recently used in combination with Cre-loxP. To enable more specific cell lineage tracing and ablation through dual recombinase activity, we generated two mouse lines that render Dre- or Dre+Cre-mediated recombination to excise a stop codon sequence that prevents the expression of diphtheria toxin receptor (DTR) knocked into the ubiquitously expressed and safe Rosa26 locus. Using different Dre- and Cre-expressing mouse lines, we showed that the surrogate gene reporters tdTomato and DTR were simultaneously expressed in target cells and in their descendants, and we observed efficient ablation of tdTomato+ cells after diphtheria toxin administration. These mouse lines were used to simultaneously trace and deplete the target cells of interest through the inducible expression of a reporter and DTR using dual Cre and Dre recombinases, allowing a more precise and efficient study of the role of specific cell subsets within a heterogeneous population in pathophysiological conditions in vivo.

An Active Image-Based Mobile Food Record Is Feasible for Capturing Eating Occasions among Infants Ages 3-12 Months Old in Hawai'i.

The ability to comprehensively assess the diet of infants is essential for monitoring adequate growth; however, it is challenging to assess dietary intake with a high level of accuracy. Infants rely on surrogate reporting by caregivers. This study aimed to determine if surrogate reporters (e.g., caregivers) could use an image-based mobile food record adapted (baby mFR) to record infants’ eating occasions, and via caregiver feedback, could assess the usability and feasibility of the baby mFR in recording infants’ diets. This was a cross-sectional study in which surrogate reporters (e.g., caregivers) recorded all food and beverage intake (including human milk) of the infant over a 4-day period. Trained research staff evaluated all images submitted during data collection for different indicators of quality. All surrogate reporters were asked to complete a usability questionnaire at the end of the 4-day data collection period. Basic descriptive analyses were performed on the infants 3–12 months of age (n = 70). A total of 91% (n = 64) of surrogate reporters used the baby mFR to record their infants’ eating occasions. The mean number of images submitted daily per participant via the mFR was 4.2 (SD 0.2). A majority of submitted images contained the fiducial marker and the food and/or beverage was completely visible. The mFR was found to be easy to use; however, suggestions were provided to increase utility of the application such as the inclusion of a bottle button and reminders. An image-based dietary assessment method using a mobile app was found to be feasible for surrogate reporters to record an infant’s food and beverage intake throughout the day.

Identification of splice regulators of fibronectin-EIIIA and EIIIB by direct measurement of exon usage in a flow-cytometry based CRISPR screen

The extracellular matrix protein fibronectin (FN) is alternatively spliced in a variety of inflammatory conditions, resulting in increased inclusion of alternative exons EIIIA and EIIIB. Inclusion of these exons affects fibril formation, fibrosis, and inflammation. To define upstream regulators of alternative splicing in FN, we have developed an in vitro flow-cytometry based assay, using RNA-binding probes to determine alternative exon inclusion level in aortic endothelial cells. This approach allows us to detect exon inclusion in the primary transcripts themselves, rather than in surrogate splicing reporters. We validated this assay in cells with and without FN-EIIIA and -EIIIB expression. In a small-scale CRISPR KO screen of candidate regulatory splice factors, we successfully detected known regulators of EIIIA and EIIIB splicing, and detected several novel regulators. Finally, we show the potential in this approach to broadly interrogate upstream signaling pathways in aortic endothelial cells with a genome-wide CRISPR-KO screen, implicating the TNFalpha and RIG-I-like signaling pathways and genes involved in the regulation of fibrotic responses. Thus, we provide a novel means to screen the regulation of splicing of endogenous transcripts, and predict novel pathways in the regulation of FN-EIIIA inclusion.

Analysis of the advantages of cis reporters in optimized FACS-Gal.

Flow cytometry is a powerful multiparametric technology, widely used for the identification, quantification, and isolation of defined populations of cells based on the expression of target proteins. It also allows for the use of surrogate reporters, either enzymatic or fluorescent, to indirectly monitor the expression of these target proteins. In this work, we optimised the dissociation protocol for the detection of the enzymatic reporter LacZ using the FACS-Gal detection system with the fluorogenic substrate FDG to compare cis- versus trans-positioned reporters efficiency. Particularly, for the FACS-Gal optimization, we studied lung and haematopoietic tissues, focusing on cell recovery, viability, FDG loading conditions and distribution of cellular populations. Reporter genes such as LacZ can be placed together with the gene of interest in the same polycistronic mRNA (in cis), or in independent alleles (in trans), which can strongly affect the correlation with the reporter readout. To address this issue, we generated a mouse model containing both types of reporters for the same gene, and compared them. Our results clearly indicate that trans-positioned reporters can be misleading, and that using a reporter gene in cis rather than trans is a much more specific method to sort for cells undergoing Cre-mediated recombination. © 2017 International Society for Advancement of Cytometry.

A stable but reversible integrated surrogate reporter for assaying CRISPR/Cas9-stimulated homology-directed repair

The discovery and application of CRISPR/Cas9 technology for genome editing has greatly accelerated targeted mutagenesis in a variety of organisms. CRISPR/Cas9-mediated site-specific cleavage is typically exploited for the generation of insertions or deletions (indels) after aberrant dsDNA repair via the endogenous non-homology end-joining (NHEJ) pathway or, alternatively, for enhancing homology-directed repair to facilitate the generation of a specific mutation (or "knock-in"). However, there is a need for efficient cellular assays that can measure Cas9/guide RNA activity. Reliable methods for enriching and identifying desired mutants are also lacking. Here we describe a method using the Piggybac transposon for stable genomic integration of an H2B-GFP reporter or a hygromycin resistance gene for assaying Cas9 target cleavage and homology-directed repair. The H2B-GFP fusion protein provides increased stability and an obvious pattern of nuclear localization. This method, called SRIRACCHA (i.e. a stable, but reversible, integrated reporter for assaying CRISPR/Cas-stimulated HDR activity), enables the enrichment of mutants via selection of GFP-positive or hygromycin-resistant mammalian cells (immortalized or non-immortalized) as a surrogate for the modification of the endogenous target site. Currently available hyperactive Piggybac transposase mutants allow both delivery and removal of the surrogate reporters, with minimal risk of generating undesirable mutations. This assay permits rapid screening for efficient guide RNAs and the accelerated identification of mutant clones and is applicable to many cell types. We foresee the utility of this approach in contexts in which the maintenance of genomic integrity is essential, for example, when engineering cells for therapeutic purposes.

Enhanced CRISPR/Cas9-mediated biallelic genome targeting with dual surrogate reporter-integrated donors.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system has recently emerged as a simple, yet powerful genome engineering tool, which has been widely used for genome modification in various organisms and cell types. However, screening biallelic genome-modified cells is often time-consuming and technically challenging. In this study, we incorporated two different surrogate reporter cassettes into paired donor plasmids, which were used as both the surrogate reporters and the knock-in donors. By applying our dual surrogate reporter-integrated donor system, we demonstrate high frequency of CRISPR/Cas9-mediated biallelic genome integration in both human HEK293T and porcine PK15 cells (34.09% and 18.18%, respectively). Our work provides a powerful genetic tool for assisting the selection and enrichment of cells with targeted biallelic genome modification.

Comparison of surrogate reporter systems for enrichment of cells with mutations induced by genome editors

Genome editors are powerful tools that allow modification of the nuclear DNA in eukaryotic cells both in vitro and in vivo. In vitro modified cells are often phenotypically indistinguishable from unmodified cells, hampering their isolation for analysis. Episomal reporters encoding fluorescent proteins can be used for enrichment of modified cells by flow cytometry. Here we compare two surrogate reporters, RGS and SSA, for the enrichment of porcine embryonic fibroblasts containing mutations induced by ZFNs or CRISPR/Cas9. Both systems were effective for enrichment of edited porcine cells with the RGS reporter proving more effective than the SSA reporter. We noted a higher-fold enrichment when editing events were induced by Cas9 compared to those induced by ZFNs, allowing selection at frequencies as high as 70%.

Efficient fdCas9 Synthetic Endonuclease with Improved Specificity for Precise Genome Engineering.

The Cas9 endonuclease is used for genome editing applications in diverse eukaryotic species. A high frequency of off-target activity has been reported in many cell types, limiting its applications to genome engineering, especially in genomic medicine. Here, we generated a synthetic chimeric protein between the catalytic domain of the FokI endonuclease and the catalytically inactive Cas9 protein (fdCas9). A pair of guide RNAs (gRNAs) that bind to sense and antisense strands with a defined spacer sequence range can be used to form a catalytically active dimeric fdCas9 protein and generate double-strand breaks (DSBs) within the spacer sequence. Our data demonstrate an improved catalytic activity of the fdCas9 endonuclease, with a spacer range of 15–39 nucleotides, on surrogate reporters and genomic targets. Furthermore, we observed no detectable fdCas9 activity at known Cas9 off-target sites. Taken together, our data suggest that the fdCas9 endonuclease variant is a superior platform for genome editing applications in eukaryotic systems including mammalian cells.

Recent developments and clinical studies utilizing engineered zinc finger nuclease technology.

Efficient methods for creating targeted genetic modifications have long been sought for the investigation of gene function and the development of therapeutic modalities for various diseases, including genetic disorders. Although such modifications are possible using homologous recombination, the efficiency is extremely low. Zinc finger nucleases (ZFNs) are custom-designed artificial nucleases that make double-strand breaks at specific sequences, enabling efficient targeted genetic modifications such as corrections, additions, gene knockouts and structural variations. ZFNs are composed of two domains: (i) a DNA-binding domain comprised of zinc finger modules and (ii) the FokI nuclease domain that cleaves the DNA strand. Over 17 years after ZFNs were initially developed, a number of improvements have been made. Here, we will review the developments and future perspectives of ZFN technology. For example, ZFN activity and specificity have been significantly enhanced by modifying the DNA-binding domain and FokI cleavage domain. Advances in culture methods, such as the application of a cold shock and the use of small molecules that affect ZFN stability, have also increased ZFN activity. Furthermore, ZFN-induced mutant cells can be enriched using episomal surrogate reporters. Additionally, we discuss several ongoing clinical studies that are based on ZFN-mediated genome editing in humans. These breakthroughs have substantially facilitated the use of ZFNs in research, medicine and biotechnology.

Enrichment of cells with TALEN-induced mutations using surrogate reporters

Targeted gene knockout using engineered nucleases such as transcription activator like-effector nucleases (TALENs) is a gold standard for investigating the functions of a gene of interest. Although most TALENs can cleave chromosomal DNA efficiently, the activities of designed TALENs are not always high enough to allow the efficient derivation of cells containing TALEN-driven mutations. Thus, simple methods to enrich cells containing TALEN-directed mutations would facilitate the use of TALENs. Here we describe the enrichment of such cells using surrogate episomal reporters coupled with flow cytometric sorting, magnetic separation, or hygromycin selection.

Surrogate reporter-based enrichment of cells containing RNA-guided Cas9 nuclease-induced mutations

RNA-guided endonucleases (RGENs), which are based on the clustered, regularly interspaced, short palindromic repeat (CRISPR)-CRISPR-associated (Cas) system, have recently emerged as a simple and efficient tool for genome editing. However, the activities of prepared RGENs are sometimes low, hampering the generation of cells containing RGEN-induced mutations. Here we report efficient methods to enrich cells containing RGEN-induced mutations by using surrogate reporters. HEK293T cells are cotransfected with the reporter plasmid, a plasmid encoding Cas9 and a plasmid encoding crRNA and tracrRNA, and subjected to flow cytometric sorting, magnetic separation or hygromycin selection. The selected cell populations are highly enriched with cells containing RGEN-induced mutations, by a factor of up to 11-fold as compared with the unselected population. The fold enrichment tends to be high when RGEN activity is low. We envision that these reporters will facilitate the use of RGEN in a wide range of biomedical research.

Production of CMAH Knockout Preimplantation Embryos Derived From Immortalized Porcine Cells Via TALE Nucleases

Although noncancerous immortalized cell lines have been developed by introducing genes into human and murine somatic cells, such cell lines have not been available in large domesticated animals like pigs. For immortalizing porcine cells, primary porcine fetal fibroblasts were isolated and cultured using the human telomerase reverse transcriptase (hTERT) gene. After selecting cells with neomycin for 2 weeks, outgrowing colonized cells were picked up and subcultured for expansion. Immortalized cells were cultured for more than 9 months without changing their doubling time (~24 hours) or their diameter (< 20 µm) while control cells became replicatively senescent during the same period. Even a single cell expanded to confluence in 100 mm dishes. Furthermore, to knockout the CMAH gene, designed plasmids encoding a transcription activator-like effector nuclease (TALENs) pairs were transfected into the immortalized cells. Each single colony was analyzed by the mutation-sensitive T7 endonuclease I assay, fluorescent PCR, and dideoxy sequencing to obtain three independent clonal populations of cells that contained biallelic modifications. One CMAH knockout clone was chosen and used for somatic cell nuclear transfer. Cloned embryos developed to the blastocyst stage. In conclusion, we demonstrated that immortalized porcine fibroblasts were successfully established using the human hTERT gene, and the TALENs enabled biallelic gene disruptions in these immortalized cells.

Barcoding cells using cell-surface programmable DNA-binding domains

We develop here a novel approach to barcode large numbers of cells through cell-surface expression of programmable zinc-finger DNA-binding domains (sZFs). We show sZFs enable double-stranded DNA to sequence-specifically label living cells, and also develop a sequential tagging approach to in situ image >3 cell types using just 3 fluorophores. Finally we demonstrate their broad versatility through ability to serve as surrogate reporters and facilitate selective cell capture and targeting.

Magnetic Separation and Antibiotics Selection Enable Enrichment of Cells with ZFN/TALEN-Induced Mutations

The ability to enrich cells with targeted mutations greatly facilitates the process of using engineered nucleases, including zinc-finger nucleases and transcription activator-like effector nucleases, to construct such cells. We previously used surrogate reporters to enrich cells containing nuclease-induced mutations via flow cytometry. This method is, however, limited by the availability of flow cytometers. Furthermore, sorted cells occasionally fail to form colonies after exposure to a strong laser and hydrostatic pressure. Here we describe two different types of novel reporters that enable mutant cell enrichment without the use of flow cytometers. We designed reporters that express H-2Kk, a surface antigen, and the hygromycin resistance protein (HygroR), respectively, when insertions or deletions are generated at the target sequences by the activity of engineered nucleases. After cotransfection of these reporters and the engineered nuclease-encoding plasmids, H-2Kk- and HygroR-expressing cells were isolated using magnetic separation and hygromycin treatment, respectively. We found that mutant cells were drastically enriched in the isolated cells, suggesting that these two reporters enable efficient enrichment of mutants. We propose that these two reporters will greatly facilitate the use of engineered nucleases in a wider range of biomedical research.

Surrogate reporters for enrichment of cells with nuclease-induced mutations

Zinc-finger nucleases (ZFNs) and TAL-effector nucleases (TALENs) are powerful tools for creating genetic modifications in eukaryotic cells and organisms. But wild-type and mutant cells that contain genetic modifications induced by these programmable nucleases are often phenotypically indistinguishable, hampering isolation of mutant cells. Here we show that transiently transfected episomal reporters encoding fluorescent proteins can be used as surrogate genes for the efficient enrichment of endogenous gene-modified cells by flow cytometry.

Workshop 2: The use of surrogate reporters in the assessment of dietary intake

Workshop 2: The use of surrogate reporters in the assessment of dietary intake

Quantitative dissection of clone-specific growth rates in cultured malaria parasites

Measurement of parasite proliferation in cultured red blood cells underpins many facets of malaria research, from drug sensitivity assays to assessing the impact of experimentally altered genes on parasite growth, virulence and fitness. Pioneering efforts to grow Plasmodium falciparum in cultured red blood cells revolutionised malaria research and spurred the development of semi-high-throughput growth assays using radio-labelled hypoxanthine (Hx), an essential nucleic acid precursor, as a reporter of whole-cycle proliferation [Trager, W., Jensen, J.B., 1976. Human malaria parasites in continuous culture. Science 193, 673–675; Desjardins, R.E., Canfield, C.J., Haynes, J.D., Chulay, J.D., 1979. Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob. Agents Chemother. 16, 710–718]. The isotopic Hx assay remains the standard quantitative growth assay with which newer non-radioactive procedures based on fluorescent DNA dyes or ELISA are compared. All of these readouts are surrogate reporters of changes in bulk parasitemias, reflecting proliferation over entire asexual reproductive cycles. While quantitatively robust and amenable to semi-high-throughput applications, these methods are blind to the underlying developmental and cellular events of growth in human red blood cells. Modern whole-genome tools including gene knockouts, mutagenesis and small molecule screens promise to reveal much about basic parasite biology; however methods to precisely quantify the within-cycle growth process are needed. Here we elaborate on the classical growth index, i.e. changes in parasitemia, by quantifying sub-phenotypes of a rapid proliferator, the multi-drug resistant clone Dd2, and a standard wild-type clone, HB3. These data illustrate differences in cycle duration, merozoite production, and invasion rate and efficiency that underpin Dd2’s average 2-fold proliferation advantage over HB3 per erythrocytic cycle. The ability to refine growth phenotypes will inform the search for molecular determinants of differential parasite growth rates and broaden our understanding of killing mechanisms and cellular targets of antimalarial drugs.

Depression in Alzheimer's Disease Patients: Caregivers as Surrogate Reporters

To evaluate the use of caregivers as surrogate reporters of depressive symptoms in Alzheimer's disease (AD) patients on five depression measures. Scale characteristics, including means, ranges, internal consistency, sensitivity, and item content of modified self-report questionnaire and interview depression measures were compared. Seventy-six patients, diagnosed with both depression and AD, and their family caregivers participated in this investigation. All subjects were community-residing participants in a treatment outcome investigation of behavioral treatment for depression. A Schedule for Affective Disorders and Schizophrenia (SADS) interview was completed on all subjects to establish a diagnosis of depression. Caregivers then completed three additional questionnaire measures about their patient: the Beck Depression Inventory (BDI), the Geriatric Depression Scale (GDS), and the Center for Epidemiological Studies-Depression Scale (CESD), all modified to provide a surrogate report of their patient's depressive symptoms. In addition, two interview measures were completed based on interviews with the caregiver and patient: the Hamilton Depression Rating Scale (HDRS) and the Cornell Scale for Depression in Dementia (CSDD). Mean scores for this sample were above the recommended cutoff score for mild levels of depression on all measures. Coefficient alpha levels were comparable to levels reported for the traditional self-report formats. Sensitivity varied among the measures, with the CSDD most sensitive and the HDRS least sensitive. Item content varied across measures, particularly between questionnaire and interview measures. Based on these results, it appears that caregivers are able to act as surrogate reporters of depression in AD patients. The modification of self-report questionnaires did not decrease their internal consistency, and they remained highly correlated with each other, supporting their validity when used in this manner. A two-step process of evaluating AD patients for depression is recommended: first, a surrogate report questionnaire completed by caregivers to screen patients, eliminating those who are unlikely to be depressed, and then a more extensive interview with those patients-and their caregivers- who appear likely to be depressed.