Parenchymal obliteration by renal masses: Functional and oncologic implications.

OBJECTIVES: Most renal tumors merely displace nephrons while others can obliterate parenchyma in an invasive manner. Substantial parenchymal volume replacement (PVR) by renal cell carcinoma (RCC) may have oncologic implications; however, studies regarding PVR remain limited. Our objective was to evaluate the oncologic implications associated with PVR using improved methodology including more accurate and objective tools. PATIENTS/METHODS: A total of 1,222 patients with non-metastatic renal tumors managed with partial nephrectomy (PN) or radical nephrectomy (RN) at Cleveland Clinic (2011-2014) with necessary studies were retrospectively evaluated. Parenchymal volume analysis via semiautomated software was used to estimate split renal function and preoperative parenchymal volumes. Using the contralateral kidney as a control, %PVR was defined: (parenchymal volumecontralateral-parenchymal volumeipsilateral) normalized by parenchymal volumecontralateral x100%. PVR was determined preoperatively and not altered by management. Patients were grouped by degree of PVR: minimal (<5%, N = 566), modest (5%-25%, N = 414), and prominent (≥25%, N = 142). Kaplan-Meier was used to evaluate survival outcomes relative to degree of PVR. Multivariable Cox-regression models evaluated predictors of recurrence-free survival (RFS). RESULTS: Of 1,122 patients, 801 (71%) were selected for PN and 321 (29%) for RN. Overall, median tumor size was 3.1 cm and 6.8 cm for PN and RN, respectively, and median follow-up was 8.6 years. Median %PVR was 15% (IQR = 6%-29%) for patients selected for RN and negligible for those selected for PN. %PVR correlated inversely with preoperative ipsilateral GFR (r = -0.49, P < 0.01) and directly with advanced pathologic stage, high tumor grade, clear cell histology, and sarcomatoid features (all P < 0.01). PVR≥25% associated with shortened recurrence-free, cancer-specific, and overall survival (all P < 0.01). Male sex, ≥pT3a, tumor grade 4, positive surgical margins, and PVR≥25% independently associated with reduced RFS (all P < 0.02). CONCLUSIONS: Obliteration of normal parenchyma by RCC substantially impacts preoperative renal function and patient selection. Our data suggests that increased PVR is primarily driven by aggressive tumor characteristics and independently associates with reduced RFS, although further studies will be needed to substantiate our findings.

Limitations of Parenchymal Volume Analysis for Estimating Split Renal Function and New Baseline Glomerular Filtration Rate After Radical Nephrectomy.

PURPOSE: Accurately predicting new baseline glomerular filtration rate (NBGFR) after radical nephrectomy (RN) can improve counseling about RN vs partial nephrectomy. Split renal function (SRF)-based models are optimal, and differential parenchymal volume analysis (PVA) is more accurate than nuclear renal scans (NRS) for this purpose. However, there are minimal data regarding the limitations of PVA. Our objective was to identify patient-/tumor-related factors associated with PVA inaccuracy. MATERIALS AND METHODS: Five hundred and ninety-eight RN patients (2006-2021) with preoperative CT/MRI were retrospectively analyzed, with 235 also having NRS. Our SRF-based model to predict NBGFR was: 1.25 × (GlobalGFRPre-RN × SRFContralateral), where GFR indicates glomerular filtration rate, with SRF determined by PVA or NRS, and with 1.25 representing the median renal functional compensation in adults. Accuracy of predicted NBGFR within 15% of observed was evaluated in various patient/tumor cohorts using multivariable logistic regression analysis. RESULTS: PVA and NRS accuracy were 73%/52% overall, and 71%/52% in patients with both studies (n = 235, P < .001), respectively. PVA inaccuracy independently associated with pyelonephritis, hydronephrosis, renal vein thrombosis, and infiltrative features (all P < .03). Ipsilateral hydronephrosis and renal vein thrombosis associated with PVA underprediction, while contralateral hydronephrosis and increased age associated with PVA overprediction (all P < .01). NRS inaccuracy was more common and did not associate with any of these conditions. Even among cohorts where PVA inaccuracy was observed (22% of our patients), there was no significant difference in the accuracies of NRS- and PVA-based predictions. CONCLUSIONS: PVA was more accurate for predicting NBGFR after RN than NRS. Inaccuracy of PVA correlated with factors that distort the parenchymal volume/function relationship or alter renal functional compensation. NRS inaccuracy was more common and unpredictable, likely reflecting the inherent inaccuracy of NRS. Awareness of cohorts where PVA is less accurate can help guide clinical decision-making.

The Evolving Landscape of Male Varicocele Pathophysiology in the Era of Multi-Omics: A Narrative Review of the Current Literature.

Male-factor infertility is implicated in over half of the millions of cases of infertility worldwide, and varicoceles are the most common correctable cause of male-factor infertility. The pathophysiologic mechanism for varicoceles is complex and next-generation technologies offer promising insights into the molecular underpinnings of this condition. In this narrative review, we highlight historical and contemporary paradigms associated with varicoceles, with an emphasis on the biological underpinnings of this disease. Specifically, we review the literature describing the underlying causes of varicoceles, discuss the molecular and cellular mechanisms causing pathological changes in some (but not all) men, and highlight key articles regarding the next-generation analyses (e.g., transcriptome, epigenome, proteome, and microbiome) being applied to better understand the condition and its treatment. These data demonstrate an ongoing evolution of the knowledge of varicoceles and the potential for improved personalized care in the future for men with this condition.

Parenchymal volume preservation during partial nephrectomy: improved methodology to assess impact and predictive factors.

OBJECTIVE: To rigorously evaluate the impact of the percentage of parenchymal volume preserved (PPVP) and how well the preserved parenchyma recovers from ischaemia (Recischaemia ) on functional outcomes after partial nephrectomy (PN) using an accurate and objective software-based methodology for estimating parenchymal volumes and split renal function (SRF). A secondary objective was to assess potential predictors of the PPVP. PATIENTS AND METHODS: A total of 894 PN patients with available studies (2011-2014) were evaluated. The PPVP was measured from cross-sectional imaging at ≤3 months before and 3-12 months after PN using semi-automated software. Pearson correlation evaluated relationships between continuous variables. Multivariable linear regression evaluated predictors of ipsilateral glomerular filtration rate (GFR) preserved and the PPVP. Relative-importance analysis was used to evaluate the impact of the PPVP on ipsilateral GFR preserved. Recischaemia was defined as the percentage of ipsilateral GFR preserved normalised by the PPVP. RESULTS: The median tumour size and R.E.N.A.L. nephrometry score were 3.4 cm and 7, respectively. In all, 49 patients (5.5%) had a solitary kidney. In all, 538 (60%)/251 (28%)/104 (12%) patients were managed with warm/cold/zero ischaemia, respectively. The median pre/post ipsilateral GFRs were 40/31 mL/min/1.73 m2 , and the median (interquartile range [IQR]) percentage of ipsilateral GFR preserved was 80% (71-88%). The median pre/post ipsilateral parenchymal volumes were 181/149 mL, and the median (IQR) PPVP was 84% (76-92%). In all, 330 patients (37%) had a PPVP of <80%, while only 34 (4%) had a Recischaemia of <80%. The percentage of ipsilateral GFR preserved correlated strongly with the PPVP (r = 0.83, P < 0.01) and loss of parenchymal volume accounted for 80% of the loss of ipsilateral GFR. Multivariable analysis confirmed that the PPVP was the strongest predictor of ipsilateral GFR preserved. Greater tumour size and endophytic and nearness properties of the R.E.N.A.L. nephrometry score were associated with a reduced PPVP (all P ≤ 0.01). Solitary kidney and cold ischaemia were associated with an increased PPVP (all P < 0.05). CONCLUSIONS: A reduced PPVP predominates regarding functional decline after PN, although a low Recischaemia can also contribute. Tumour-related factors strongly influence the PPVP, while surgical efforts can improve the PPVP as observed for patients with solitary kidneys.

Thermal Ablation Versus Partial Nephrectomy for cT1 Renal Mass in a Solitary Kidney: A Matched Cohort Comparative Analysis.

BACKGROUND: Nephron-sparing approaches are preferred for renal mass in a solitary kidney (RMSK), with partial nephrectomy (PN) generally prioritized. Thermal ablation (TA) also is an option for small renal masses in this setting; however, comparative functional/survival outcomes are not well-defined. METHODS: A retrospective study of 504 patients (1975-2022) with cT1 RMSK managed with PN (n = 409)/TA (n = 95) with necessary data for analysis was performed. Propensity score was used for matching patients, including age, preoperative glomerular filtration rate (GFR), tumor diameter, R.E.N.A.L. ((R)adius (tumor size as maximal diameter), (E)xophytic/endophytic properties of tumor, (N)earness of tumor deepest portion to collecting system or sinus, (A)nterior (a)/posterior (p) descriptor, and (L)ocation relative to polar lines), and comorbidities. Functional outcomes were compared, and Kaplan-Meier was used to analyze survival. RESULTS: The matched cohort included 132 patients (TA = 66/PN = 66), with median tumor diameter of 2.4 cm, R.E.N.A.L. of 6, and preoperative GFR of 52 ml/min/1.73 m2. Acute kidney injury occurred in 11%/61% in the TA/PN cohorts, respectively (p < 0.01). After recovery, median GFR preserved was 89%/83% for TA/PN, respectively (p = 0.02), and 5-year dialysis-free survival was 96% in both cohorts. Median follow-up was 53 months. Five-year recurrence-free survival (RFS) was 62%/86% in the TA/PN cohorts, respectively (p < 0.01). Five-year local recurrence (LR)-free survival was 74%/95% in the TA/PN cohorts, respectively (p < 0.01). Five-year cancer-specific survival (CSS) was 96%/98% in the TA/PN cohorts, respectively (p = 0.7). Local recurrence was observed in nine of 36 (25%) and five of 30 (17%) patients managed with laparoscopic versus percutaneous TA, respectively. For TA with LR (n = 14), nine patients presented with multifocality and/or cT1b tumors. Twelve LR were managed with salvage TA, and seven remained cancer-free, while five developed systemic recurrence, three with concomitant LR. CONCLUSIONS: Functional outcomes for TA for RMSK were improved compared with PN. Local recurrence was more common after TA and often was associated with the laparoscopic approach, multifocality, and large tumor size. Improved patient selection and greater experience with TA should improve outcomes. Salvage of LR was not always possible. Partial nephrectomy remains the reference standard for RMSK.

Practical Prediction of New Baseline Renal Function After Partial Nephrectomy.

BACKGROUND: Partial nephrectomy (PN) is generally preferred for localized renal masses due to strong functional outcomes. Accurate prediction of new baseline glomerular filtration rate (NBGFR) after PN may facilitate preoperative counseling because NBGFR may affect long-term survival, particularly for patients with preoperative chronic kidney disease. Methods for predicting parenchymal volume preservation, and by extension NBGFR, have been proposed, including those based on contact surface area (CSA) or direct measurement of tissue likely to be excised/devascularized during PN. We previously reported that presuming 89% of global GFR preservation (the median value saved from previous, independent analyses) is as accurate as the more subjective/labor-intensive CSA and direct measurement approaches. More recently, several promising complex/multivariable predictive algorithms have been published, which typically include tumor, patient, and surgical factors. In this study, we compare our conceptually simple approach (NBGFRPost-PN = 0.90 × GFRPre-PN) with these sophisticated algorithms, presuming that an even 90% of the global GFR is saved with each PN. PATIENTS AND METHODS: A total of 631 patients with bilateral kidneys who underwent PN at Cleveland Clinic (2012-2014) for localized renal masses with available preoperative/postoperative GFR were analyzed. NBGFR was defined as the final GFR 3-12 months post-PN. Predictive accuracies were assessed from correlation coefficients (r) and mean squared errors (MSE). RESULTS: Our conceptually simple approach based on uniform 90% functional preservation had equivalent r values when compared with complex, multivariable models, and had the lowest degree of error when predicting NBGFR post-PN. CONCLUSIONS: Our simple formula performs equally well as complex algorithms when predicting NBGFR after PN. Strong anchoring by preoperative GFR and minimal functional loss (≈ 10%) with the typical PN likely account for these observations. This formula is practical and can facilitate counseling about expected postoperative functional outcomes after PN.

The effect of different types of oral or intravenous corticosteroids on capillary blood glucose levels in hospitalized inpatients with and without diabetes.

PURPOSE: This study investigated: (1) the type of corticosteroid associated with the greatest degree of hyperglycemia, assessed using bedside capillary blood glucose monitoring, in hospitalized patients; and (2) the pattern of hyperglycemia throughout the day with the use of each type of corticosteroid. METHODS: This single-center, retrospective study used data from 964 adult inpatients receiving oral or IV corticosteroids. Data on capillary blood glucose concentrations and time taken over 7 days were collected. A mixed model for repeated measures was applied to investigate changes in glucose concentration over time with the use of four different corticosteroids. An autoregressive covariance structure was used to model correlations between repeated measurements. FINDINGS: Across all 7 days, the mean blood glucose concentration was greater with dexamethasone compared to that with hydrocortisone (mean difference, 16.6 mg/dL [95% CI, 8.1-24.8] [0.92 mmol/L (95% CI, 0.45-1.38)]) or prednisolone (mean difference, 20.0 mg/dL [95% CI, 14.2-25.7] [1.11 mmol/L (95% CI, 0.79-1.43)]). The mean blood glucose concentration was greater with methylprednisolone compared to that with hydrocortisone (mean difference, 23.9 mg/dL [95% CI, 11.3-36.4] [1.33 mmol/L (95% CI, 0.63-2.02)]), and with methylprednisolone versus prednisolone (mean difference, 27.4 mg/dL [95% CI, 16.4-38.3] [1.52 mmol/L (95% CI, 0.91-2.13)]). There were no significant differences in the patterns of hyperglycemia at six time points of the day with each type of corticosteroid. IMPLICATIONS: Treatment with oral or IV dexamethasone or methylprednisolone was associated with greater hyperglycemia in comparison to prednisolone and hydrocortisone. More vigorous monitoring and intervention, when necessary, are suggested in adult inpatients receiving corticosteroids, in particular dexamethasone and methylprednisolone.

Functional recovery after partial nephrectomy in a solitary kidney.

OBJECTIVES: Partial nephrectomy (PN) is the reference standard for renal mass in a solitary kidney (RMSK), although factors determining functional recovery in this setting remain poorly defined. PATIENTS/METHODS: Single center, retrospective analysis of 841 RMSK patients (1975-2022) managed with PN with functional data, including 361/435/45 with cold/warm/zero ischemia, respectively. A total of 155 of these patients also had necessary studies for detailed analysis of parenchymal volume preserved. Acute kidney injury (AKI) was classified by RIFLE (Risk/Injury/Failure/Loss/Endstage). Recovery-from-ischemia (Rec-Ischemia) was defined as glomerular filtration rate (GFR) saved normalized by parenchymal volume saved. Logistic regression identified predictive factors for AKI and predictors of Rec-Ischemia were analyzed by multivariable linear regression. RESULTS: Overall, median preoperative GFR was 56.7 ml/min/1.73m2 and new-baseline and 5-year GFRs were 43.1 and 44.5 ml/min/1.73m2, respectively. Median follow-up was 55 months; 5-year dialysis-free survival was 97%. In the detailed analysis cohort, a primary focus of this study, median warm (n = 70)/cold (n = 85) ischemia times were 25/34 minutes, respectively; and median preoperative, new-baseline and 5-year GFRs were 57.8, 45.0, and 41.7 ml/min/1.73m2, respectively. Functional recovery correlated strongly with parenchymal volume preserved (r = 0.84, p < 0.001). Parenchymal volume loss accounted for 69% of the total median GFR decline associated with PN, leaving only 3 to 4 ml/min/1.73m2 attributed to ischemia and other factors. AKI occurred in 52% of patients and the only independent predictor of AKI was ischemia time. Independent predictors of reduced Rec-Ischemia were increased age, warm ischemia, and AKI. CONCLUSION: The main determinant of functional recovery after PN in RMSK is parenchymal volume preservation. Type/duration of ischemia, AKI, and age also correlated, although altogether their contributions were less impactful. Our findings suggest multiple opportunities for optimizing functional outcomes although preservation of parenchymal volume remains predominant. Long-term function generally remains stable with dialysis only occasionally required.

Selective Use of Neoadjuvant Targeted Therapy Is Associated with Greater Achievement of Partial Nephrectomy for High-complexity Renal Masses in a Solitary Kidney.

Background: Partial nephrectomy (PN) is preferred for a renal mass in a solitary kidney (RMSK), although tumors with high complexity can be challenging. Objective: To evaluate the evolution of RMSK management with a focus on achievement of PN. Design setting and participants: Patients with nonmetastatic RMSK (n = 499) were retrospectively reviewed; 133 had high tumor complexity, including 80 in the pre-tyrosine kinase inhibitor (TKI) era (1999-2008) and 53 in the TKI era (2009-2022). After 2009, 23/53 patients received neoadjuvant TKI and 30/53 had immediate-surgery. Outcome measurements and statistical analysis: Functional outcomes, adverse events and complications, dialysis-free survival, and recurrence-free survival (RFS) were the measures evaluated. Mann-Whitney and χ2 tests were used to compare cohorts, and the log-rank test was applied for survival analyses. Results and limitations: Overall, the median RENAL score was 10 and the median tumor diameter was 5.2 cm. Demographic characteristics, tumor diameter, and RENAL scores were similar between the pre-TKI-era and TKI-era groups. In the TKI era, 23/53 patients (43%) with clear-cell histology were selected for neoadjuvant TKI. These 23 patients had a greater median tumor diameter (7.1 vs 4.4 cm; p = 0.02) and RENAL score (11 vs 10; p = 0.07). After TKI treatment, the median tumor diameter decreased to 5.6 cm and the RENAL score to 9, and tumor volume was reduced by 59% (all p < 0.05). PN was accomplished in 21/23 (91%) the TKI-treated cases and in 27/30 (90%) of the immediate-surgery cases (2009-2022). PN was only accomplished in 52/80 (65%) of the patients from the pre-TKI era (p < 0.01). The 5-yr dialysis-free survival rate was 59% in the pre-TKI-era group and 91% in the TKI-era group. The 5-yr RFS rate was lower in the TKI-era group (59% vs 74%; p = 0.21), which was mostly related to more aggressive tumor biology, as reflected by a predominance of systemic rather than local recurrences. Conclusions: Management of RMSK with high tumor complexity is challenging. Selective use of TKI therapy was associated with greater use of PN, although a randomized study is needed. RFS mostly reflected aggressive tumor biology rather than failure of local management. Patient summary: For complex kidney tumors in patients with a single kidney, management is challenging. Use of drugs called tyrosine kinase inhibitors before surgery was associated with reductions in tumor size and greater ability to achieve partial kidney removal for cancer control. Most recurrences were metastatic, which reflects aggressive tumor biology rather than failure of surgery.

Parenchymal volume analysis to assess longitudinal functional decline following partial nephrectomy.

OBJECTIVE: To identify factors associated with longitudinal ipsilateral functional decline after partial nephrectomy (PN). PATIENTS AND METHODS: Of 1140 patients managed with PN (2012-2014), 349 (31%) had imaging/serum creatinine levels pre-PN, 1-12 months post-PN (new baseline), and >3 years later necessary for inclusion. Parenchymal-volume analysis was used to determine split renal function. Patients were grouped as having significant renal comorbidity (CohortSRC : diabetes mellitus with insulin-dependence or end-organ damage, refractory hypertension, or severe pre-existing chronic kidney disease) vs not having significant renal comorbidity (CohortNoSRC ) preoperatively. Multivariable regression was used to identify predictors of annual ipsilateral parenchymal atrophy and functional decline relative to new baseline values post-PN, after the kidney had healed. RESULTS: The median follow-up was 6.3 years with 87/226/36 patients having cold/warm/zero ischaemia. The median cold/warm ischaemia times were 32/22 min. Overall, the median tumour size was 3.0 cm. The preoperative glomerular filtration rate (GFR) and new baseline GFR (NBGFR) were 81 and 71 mL/min/1.73 m2 , respectively. After establishment of the NBGFR, the median loss of global and ipsilateral function was 0.7 and 0.4 mL/min/1.73 m2 /year, respectively, consistent with the natural ageing process. Overall, the median ipsilateral parenchymal atrophy was 1.2 cm3 /year and accounted for a median of 53% of the annual functional decline. Significant renal comorbidity, age, and warm ischaemia were independently associated with ipsilateral parenchymal atrophy (all P < 0.01). Significant renal comorbidity and ipsilateral parenchymal atrophy were independently associated with annual ipsilateral functional decline (both P < 0.01). Annual median ipsilateral parenchymal atrophy and functional decline were both significantly increased for CohortSRC compared to CohortNoSRC (2.8 vs 0.9 cm3 , P < 0.01 and 0.90 vs 0.30 mL/min/1.73 m2 /year, P < 0.01, respectively). CONCLUSIONS: Longitudinal renal function following PN generally follows the normal ageing process. Significant renal comorbidities, age, warm ischaemia, and ipsilateral parenchymal atrophy were the most important predictors of ipsilateral functional decline following establishment of NBGFR.

Functional recovery after partial nephrectomy: next generation analysis.

OBJECTIVES: To provide a more rigorous assessment of factors affecting functional recovery after partial nephrectomy (PN) using novel tools that allow for analysis of more patients and improved accuracy for assessment of parenchymal volume loss, thereby revealing the potential impact of secondary factors such as ischaemia. PATIENTS AND METHODS: Of 1140 patients managed with PN (2012-2014), 670 (59%) had imaging and serum creatinine levels measured before and after PN necessary for inclusion. Recovery from ischaemia was defined as the ipsilateral glomerular filtration rate (GFR) saved normalised by parenchymal volume saved. Acute kidney injury was assessed through Spectrum Score, which quantifies the degree of acute ipsilateral renal dysfunction due to exposure to ischaemia that would otherwise be masked by the contralateral kidney. Multivariable regression was used to identify predictors of Spectrum Score and Recovery from Ischaemia. RESULTS: In all, 409/189/72 patients had warm/cold/zero ischaemia, respectively, with median (interquartile range [IQR]) ischaemia times for cold and warm ischaemia of 30 (25-42) and 22 (18-28) min, respectively. The median (IQR) global preoperative GFR and new baseline GFR (NBGFR) were 78 (63-92) and 69 (54-81) mL/min/1.73 m2 , respectively. The median (IQR) ipsilateral preoperative GFR and NBGFR were 40 (33-47) and 31 (24-38) mL/min/1.73 m2 , respectively. Functional recovery correlated strongly with parenchymal volume preserved (r = 0.83, P < 0.01). The median (IQR) decline in ipsilateral GFR associated with PN was 7.8 (4.5-12) mL/min/1.73 m2 with loss of parenchyma accounting for 81% of this loss. The median (IQR) recovery from ischaemia was similar across the cold/warm/zero ischaemia groups at 96% (90%-102%), 95% (89%-101%), and 97% (91%-102%), respectively. Independent predictors of Spectrum Score were ischaemia time, tumour complexity, and preoperative global GFR. Independent predictors of recovery from ischaemia were insulin-dependent diabetes mellitus, refractory hypertension, warm ischaemia, and Spectrum Score. CONCLUSIONS: The main determinant of functional recovery after PN is parenchymal volume preservation. A more robust and rigorous evaluation allowed us to identify secondary factors including comorbidities, increased tumour complexity, and ischaemia-related factors that are also independently associated with impaired recovery, although altogether these were much less impactful.

Point of care parenchymal volume analyses to estimate split renal function and predict functional outcomes after radical nephrectomy.

Accurate prediction of new baseline GFR (NBGFR) after radical nephrectomy (RN) can inform clinical management and patient counseling whenever RN is a strong consideration. Preoperative global GFR, split renal function (SRF), and renal functional compensation (RFC) are fundamentally important for the accurate prediction of NBGFR post-RN. While SRF has traditionally been obtained from nuclear renal scans (NRS), differential parenchymal volume analysis (PVA) via software analysis may be more accurate. A simplified approach to estimate parenchymal volumes and SRF based on length/width/height measurements (LWH) has also been proposed. We compare the accuracies of these three methods for determining SRF, and, by extension, predicting NBGFR after RN. All 235 renal cancer patients managed with RN (2006-2021) with available preoperative CT/MRI and NRS, and relevant functional data were analyzed. PVA was performed on CT/MRI using semi-automated software, and LWH measurements were obtained from CT/MRI images. RFC was presumed to be 25%, and thus: Predicted NBGFR = 1.25 × Global GFRPre-RN × SRFContralateral. Predictive accuracies were assessed by mean squared error (MSE) and correlation coefficients (r). The r values for the LWH/NRS/software-derived PVA approaches were 0.72/0.71/0.86, respectively (p < 0.05). The PVA-based approach also had the most favorable MSE, which were 120/126/65, respectively (p < 0.05). Our data show that software-derived PVA provides more accurate and precise SRF estimations and predictions of NBGFR post-RN than NRS/LWH methods. Furthermore, the LWH approach is equivalent to NRS, precluding the need for NRS in most patients.

Conditional Recruitment to a DNA-Bound CRISPR-Cas Complex Using a Colocalization-Dependent Protein Switch.

To spatially control biochemical functions at specific sites within a genome, we have engineered a synthetic switch that activates when bound to its DNA target site. The system uses two CRISPR-Cas complexes to colocalize components of a de novo-designed protein switch (Co-LOCKR) to adjacent sites in the genome. Colocalization triggers a conformational change in the switch from an inactive closed state to an active open state with an exposed functional peptide. We prototype the system in yeast and demonstrate that DNA binding triggers activation of the switch, recruitment of a transcription factor, and expression of a downstream reporter gene. This DNA-triggered Co-LOCKR switch provides a platform to engineer sophisticated functions that should only be executed at a specific target site within the genome, with potential applications in a wide range of synthetic systems including epigenetic regulation, imaging, and genetic logic circuits.