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The accurate identification of mucinous pancreatic cystic lesions (PCLs) is paramount for cancer risk stratification. Cyst fluid carcinoembryonic antigen (CEA), the only routinely used test, requires high volumes and has low sensitivity. We aimed to compare the performance of two investigational small-volume biomarkers, glucose and the protease gastricsin, to CEA for PCL classification.We obtained cyst fluid samples from 81 patients with pathologically confirmed PCLs from four institutions between 2003 and 2016. Gastricsin activity was measured using an internally quenched fluorescent substrate. Glucose levels were measured with a standard glucometer. CEA levels were obtained from the medical record. Models using Classification and Regression Trees were created to predict mucinous status. Model performance was evaluated using nested cross-validation.Gastricsin activity, CEA, and glucose levels from patients with mucinous (n = 50) and nonmucinous (n = 31) PCLs were analyzed. Area under the curve (AUC) was similar for individual classifiers (gastricsin volume normalized [GVN] 0.88; gastricsin protein concentration normalized [GPN] 0.95; glucose 0.83; CEA 0.84). The combination of two classifiers did not significantly improve AUC, with CEA + GVN (0.88) performing similarly to CEA + GPN (0.95), GVN + glucose (0.87), GPN + glucose (0.95), and CEA + glucose (0.84). The three-analyte combination performed similarly to single and dual classifiers (GPN + glucose + CEA AUC 0.95; GVN + glucose + CEA AUC 0.87). After multiple comparison corrections, there were no significant differences between the individual, dual, and triple classifiers.Gastricsin and glucose performed similarly to CEA and required5% of the volume required for CEA; these classifiers may be useful in patients with limited cyst fluid. Future multicenter prospective studies are needed to validate and compare these novel small-volume biomarkers.

Disparities following traumatic injury by race/ethnicity and insurance status are well-documented. However, the relationship between limited English proficiency (LEP) and outcomes after trauma is poorly understood. This study describes the association between LEP and morbidity and mortality after traumatic injury.A retrospective cohort study was conducted of adult trauma patients admitted to a level 1 trauma center from 2012 to 2018. Morbidity (length of stay [LOS], intensive care unit admission, intensive care unit LOS, discharge destination) and in-hospital mortality for LEP and English proficient (EP) patients were compared using univariate and multivariable logistic and generalized linear models controlling for patient demographics (age, sex, race/ethnicity, insurance) and clinical characteristics (mechanism, activation level, Glasgow Coma Scale, Injury Severity Score, traumatic brain injury).Of the 13,104 patients, 16% were LEP patients. LEP languages included Chinese (44%) and Spanish (38%), and 18% categorized as "Other," including 33 languages. In multivariable models, LEP was statistically significantly associated with increased hospital LOS (P = 0.003) and increased discharge to home with home health services (P = 0.042) or to skilled nursing facility/rehabilitation (P = 0.006). Mortality rate was 7% for LEP versus 4% for EP patients (P 0.0001). In multivariable analysis, speaking an LEP language other than Chinese or Spanish was statistically significantly associated with increased mortality compared to EP (P = 0.006).Following traumatic injury, LEP patients experience increased hospital LOS and are more frequently discharged to home with home health services or to skilled nursing facilities/rehabilitation. LEP patients speaking languages other than Chinese or Spanish experience increased mortality compared to EP patients.

Objective The COVID-19 pandemic provided an opportunity for surgical residency programs to rethink their methods of evaluating and recruiting candidates. However, the past year has not been seamless, with a soaring number of applications, reports of programs and applicants having difficulty evaluating each other, and an increasingly uneven distribution of interviews among applicants. Consequently, many have called for national changes to the residency application process to address these longstanding concerns. Results Here, we review the evolving literature and advocate for the permanent adoption of visiting rotations, virtual interviews with a universal release date and data-driven attendance limits, and opportunities for in-person applicant visits. Conclusions We believe these changes leverage the strengths of each format, allow for satisfactory bidirectional evaluation, and promote principles of justice, equity, diversity, and inclusion.

The ∼30 Mb genomes of the Plasmodium parasites that cause malaria each encode ∼5000 genes, but the functions of the majority remain unknown. This is due to a paucity of functional annotation from sequence homology, which is compounded by low genetic tractability compared with many model organisms. In recent years technical breakthroughs have made forward and reverse genome-scale screens in Plasmodium possible. Furthermore, the adaptation of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-Associated protein 9 (CRISPR/Cas9) technology has dramatically improved gene editing efficiency at the single gene level. Here, we review the arrival of genetic screens in malaria parasites to analyse parasite gene function at a genome-scale and their impact on understanding parasite biology. CRISPR/Cas9 screens, which have revolutionised human and model organism research, have not yet been implemented in malaria parasites due to the need for more complex CRISPR/Cas9 gene targeting vector libraries. We therefore introduce the reader to CRISPR-based screens in the related apicomplexan Toxoplasma gondii and discuss how these approaches could be adapted to develop CRISPR/Cas9 based genome-scale genetic screens in malaria parasites. Moreover, since more than half of Plasmodium genes are required for normal asexual blood-stage reproduction, and cannot be targeted using knockout methods, we discuss how CRISPR/Cas9 could be used to scale up conditional gene knockdown approaches to systematically assign function to essential genes. Instituto de Biotecnología Fil: Ishizaki, Takahiro. Umeå University. Department of Molecular Biology; Suecia Fil: Ishizaki, Takahiro. The Laboratory for Molecular Infection Medicine Sweden (MIMS); Suecia Fil: Hernandez, Sophia. Umeå University. Department of Molecular Biology; Suecia Fil: Hernandez, Sophia. The Laboratory for Molecular Infection Medicine Sweden (MIMS); Suecia Fil: Paoletta, Martina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; Argentina Fil: Paoletta, Martina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Paoletta, Martina. Umeå University. Department of Molecular Biology; Suecia Fil: Paoletta, Martina. The Laboratory for Molecular Infection Medicine Sweden (MIMS); Suecia Fil: Sanderson, Theo. Francis Crick Institute; Reino Unido Fil: Bushell, Ellen S. C. Umeå University. Department of Molecular Biology; Suecia Fil: Bushell, Ellen S. C. The Laboratory for Molecular Infection Medicine Sweden (MIMS); Suecia

BackgroundResistant hypertension is common in patients with primary aldosteronism and in those with obstructive sleep apnea. Primary aldosteronism treatment improves sleep apnea. Despite Endocrine Society guidelines' inclusion of sleep apnea and hypertension co-diagnosis as a primary aldosteronism screening indication, the state of screening implementation is unknown.MethodsAll hypertensive adult patients with obstructive sleep apnea (n=4751) at one institution between 2012 and 2020 were compared with a control cohort without sleep apnea (n=117,815). We compared the association of primary aldosteronism diagnoses, risk factors, and screening between both groups. Patients were considered to have screening if they had a primary aldosteronism diagnosis or serum aldosterone or plasma renin activity evaluation.ResultsObstructive sleep apnea patients were predominantly men and had higher body mass index. On multivariable analysis, hypertensive sleep apnea patients had higher odds of drug-resistant hypertension (odds ratio [OR] 2.70; P < .001) and hypokalemia (OR 1.26; P < .001) independent of body mass index, sex, and number of antihypertensive medications. Overall, sleep apnea patients were more likely to be screened for primary aldosteronism (OR 1.45; P < .001); however, few patients underwent screening whether they had sleep apnea or not (pre-guideline publication 7.8% vs 4.6%; post-guidelines 3.6% vs 4.6%; P < .01). Screening among eligible sleep apnea patients remained low prior to and after guideline publication (4.4% vs 3.4%).ConclusionsObstructive sleep apnea is associated with primary aldosteronism risk factors without formal diagnosis, suggesting screening underutilization and underdiagnosis. Strategies are needed to increase screening adherence, as patients may benefit from treatment of concomitant primary aldosteronism to reduce sleep apnea severity and its associated cardiopulmonary morbidity.

Background Robotic transanal minimally invasive surgery (R-TAMIS) is an appealing alternative to transanal minimally invasive surgery (TAMIS) and transanal endoscopic microsurgery (TEM) for benign and early malignant rectal lesions that are not amenable to traditional open transanal excision. However, no studies to our knowledge have directly compared the three techniques. This study sought to compare peri-operative and pathologic outcomes of the three approaches. Methods The records of 29 consecutive patients who underwent TEM, TAMIS, or R-TAMIS at a single academic center between 2016 and 2020 were reviewed. Intra-operative details, pathological diagnosis and margins, and post-operative outcomes were recorded. The three groups were compared using chi-square and Kruskal–Wallis tests. Results Overall, 16/29 patients were women and the median age was 57 (interquartile range (IQR): 28–81). Thirteen patients underwent TEM, six had TAMIS, and 10 had R-TAMIS. BMI was lower in the R-TAMIS patients (24.7; IQR 23.8–28.7), than in TEM (29.3; IQR 19.9–30.2), and TAMIS (30.4; IQR 26.6–32.9) patients. High grade dysplasia and/or invasive cancer was more common in TAMIS (80%) and R-TAMIS (66.7%) patients than in TEM patients (41.7%). The three groups did not differ significantly in tumor type or distance from the anal verge. No R-TAMIS patients had a positive surgical margin compared to 23.1% in the TEM group and 16.7% in the TAMIS group. Length of stay (median 1 day for TEM and R-TAMIS patients, 0 days for TAMIS patients) and 30-day readmission rates (7.7% of TEM, 0% of TAMIS, 10% of R-TAMIS patients) also did not differ among the groups. Median operative time was 110 min for TEM, 105 min for TAMIS, and 76 min for R-TAMIS patients. Conclusions R-TAMIS may have several advantages over other advanced techniques for transanal excisions. R-TAMIS tended to be faster and to more often result in negative surgical margins compared to the two other techniques.

Introduction The impact of COVID-19 on surgical education has been profound, and clinical learning experiences transitioned to virtual formats. This study investigated the impact of virtual experiences created to facilitate learning during the pandemic for medical students. Methods We performed a cohort study to determine the perceived clinical preparedness for medical students enrolled in the preclinical surgery pilot course, surgical Extended Mastery Learning Rotation (EMLR), and longitudinal surgical clerkship (LC). The preclinical surgery pilot course took place before COVID-19 disruptions, and the EMLR and LC experiences took place virtually. Specialty choice was examined in the EMLR and LC cohorts. Performance on the NBME surgical assessments was analyzed among students enrolled in the traditional clerkship and pandemic-disrupted courses and compared to national data using a two-sample t-test. Results Compared to preclinical students, EMLR and LC students demonstrated improvements in their perceived surgical clerkship readiness. After the 3-week EMLR course, in the setting of completing only one-third of the clerkship year, students had an average NBME Surgical Self-Assessment Exam score of 72 (SD 12), comparable to the national average of 71 (SD 9) p = 0.33. The average shelf exam score for students (N = 24) enrolled in the traditional clerkship (block 1), prior to COVID-19, disruptions was 66 (SD 9) compared to an average score of 69 (SD 9) for the longitudinal clerkship students (N = 20) that took the shelf exam later in the year (p = 0.36). COVID-19 disruptions did not affect specialty choice. All LC students have decided on a specialty; 50% nonsurgical and 50% surgical. From the EMLR cohort, 36% and 38% plan to pursue surgical and nonsurgical specialties, respectively, with 26% still undecided. Conclusions Courses were well-liked and will be implemented in future clerkships. Surgical educators demonstrated flexibility and creativity in the development of the EMLR. Despite COVID-19 disruptions, medical students made progress in their clinical skills and foundational science knowledge. COVID-19 disruptions did not appear to impact specialty choice.

IntroductionThis study describes perceived knowledge gaps of third-year medical students after participating in a virtual surgical didactic rotation (EMLR) and shortened in-person surgery rotation during the COVID-19 Pandemic.MethodsOpen-ended and Likert questions were administered at the end of the virtual rotation and inperson-surgical rotation to medical students. Three blinded coders identified themes by semantic analysis.Results82 students (51% of all MS3s) participated in the EMLR. Semantic analysis revealed gaps in perioperative management (Post-EMLR:18.4%, Post-Inpatient:26.5%), anatomy (Post-EMLR:8.2%, PostInpatient:26.5%). and surgical skills (Post-EMLR: 43.0%, Post-Inpatient: 44.1%). Students also described gaps related to OR etiquette (Post-EMLR: 12.2%, Post-Inpatient: 8.8%) and team dynamics/the hidden curriculum (Post- Inpatient:26.6%). There was a significant improvement in perceived confidence to perform inpatient tasks after completing the inpatient clinical experience (p≤0.01).ConclusionVirtual interactive didactics for cognitive skills development cannot replace a full clinical surgical experience for third-year medical students. Future curricula should address perceived gaps.

Juvenile open angle glaucoma (JOAG) is a severe type of glaucoma with childhood onset and dominant inheritance. Using exome sequencing we identified 3 independent families from the Philippines with rare EFEMP1 variants (c.238A>T, p.Asn80Tyr; c.1480T>C p.Ter494Glnext*29; and c.1429C>T, p.Arg477Cysteine ) co-segregating with disease. Affected variant carriers (N= 34) exhibited severe disease with average age of onset of 16 years and with 76% developing blindness. To investigate functional effects, we transfected COS7 cells with vectors expressing the three novel EFEMP1 variants and showed that all three variants found in JOAG patients caused significant intracellular protein aggregation and retention compared to wild type and also compared to EFEMP1 variants associated with other ocular phenotypes including an early-onset form of macular degeneration, Malattia Leventinese/Doyne’s Honeycomb retinal dystrophy. These results suggest that rare EFEMP1 coding variants can cause JOAG through a mechanism involving protein aggregation and retention, and that the extent of intracellular retention correlates with disease phenotype. This is the first report of EFEMP1 variants causing JOAG, expanding the EFEMP1 disease spectrum. Our results suggest that EFEMP1 mutations appear to be a relatively common cause of JOAG in Filipino families, an ethnically diverse population.

The Association for Surgical Education formed an ad-hoc research group to evaluate the quality and methodology of the current literature on virtual surgical education. Using SIGN methodology, evidence-based guidelines were developed for utilizing virtual surgical education within the cognitive and curricula, psychomotor, and faculty development domains. Objective To identify, categorize, and evaluate the quality of literature, and to provide evidence-based guidelines on virtual surgical education within the cognitive and curricula, psychomotor, and faculty development domains. Summary background data During the COVID-19 Pandemic, utilizing virtual learning modalities is expanding rapidly. While the innovative methods must be considered to bridge the surgical education gap, a framework is needed to avoid expansion of virtual education without proper supporting evidence in some areas. Methods The Association for Surgical Education formed an ad-hoc research group to evaluate the quality and methodology of the current literature on virtual education and to build evidence-based guidelines by utilizing the Scottish Intercollegiate Guideline Network (SIGN) methodology. We identified PICO-style questions, conducted systematic literature reviews using PubMed, Embase, and ERIC databases. Then we formulated evidence-based recommendations, assessed the quality of evidence using GRADE, NOS-E and Kirkpatrick ratings, and conducted Delphi consensus to validate the recommendations. Results Eleven PICO-style questions were designed by the expert committees. After screening 4723 articles by the review committee, 241 articles met inclusion criteria for full article reviews, and 166 studies were included and categorized into three domains: cognition and curricula (n = 92), psychomotor, (n = 119) and faculty development (n = 119). Sixteen evidence-based recommendations were formulated and validated by an external expert panel. Conclusion The evidence-based guidelines developed using SIGN methodology, provide a set of recommendations for surgical training societies, training programs, and educators on utilizing virtual surgical education and highlights the area of needs for further investigation.

The hidden curriculum of unspoken professional expectations negatively impacts medical student interest in surgery. Medical student mentorship and early surgical exposure have been shown to demystify the hidden curriculum. Although residents and faculty play a vital role, near-peer mentorship may aid in uncovering the hidden curriculum and promoting medical student interest in surgery, especially for those learners who are underrepresented in medicine. We developed and implemented a formalized near-peer mentorship program composed of quarterly small group Surgical Peer Teacher led lessons and one-on-one Surgical Support Team mentorship meetings covering surgical curriculum topics for medical students at an academic medical school. This structured near-peer mentorship model provides a mechanism to demystify surgical culture, increase early access to surgical mentorship, and develop mentorship skills amongst students. This program aims to uncover the surgical hidden curriculum to improve surgical career support and interest among medical students with less exposure and access to physician role models. This longitudinal mentorship model is student-run and can be easily adapted to enhance existing support models at medical schools. Future studies will evaluate utilization, impact on surgical specialty interest, and efficacy in demystifying the surgical hidden curriculum.

Race/ethnicity has been strongly associated with substance use testing but little is known about this association in injured patients. We sought to identify trends and associations between race/ethnicity and urine toxicology (UTox) or Blood Alcohol Concentration (BAC) testing in a diverse population after trauma.We conducted a retrospective cross-sectional study of adult trauma patients admitted to a single Level-1 trauma center from 2012 to 2019. The prevalence of substance use testing was evaluated over time and analyzed using a multivariable logistic regression, with a subgroup analysis to evaluate the interaction of English language proficiency with race/ethnicity in the association of substance use testing.A total of 15,556 patients (40% White, 13% Black, 24% Latinx, 20% Asian, and 3% Native or Unknown) were included. BAC testing was done in 63.2% of all patients and UTox testing was done in 39.2%. The prevalence of substance use testing increased over time across all racial/ethnic groups. After adjustment, Latinx patients had higher odds of receiving a BAC test and Black patients had higher odds of receiving a UTox test (P 0.001 and P 0.001, respectively) compared to White patients. Asian patients had decreased odds of undergoing a UTox or BAC test compared to White patients (P 0.001 and P 0.001, respectively). Patients with English proficiency had higher odds of undergoing substance use testing compared to those with limited English proficiency (P 0.001).Despite an increase in substance use testing over time, inequitable testing remained among racial/ethnic minorities. More work is needed to combat racial/ethnic disparities in substance use testing.

Background Although death from drug overdose is a leading cause of injury-related death in the United States, its incidence after traumatic incident is unknown. Moreover, little is known about related risk factors. We sought to determine the incidence and characteristics of and risk factors for trauma patients suffering death by acute drug poisoning (“overdose”) after hospitalization for a traumatic incident. Methods We conducted a retrospective chart review of all admitted trauma patients ≥ 18 y of age at the only level-1 trauma center in our region from 2012 to 2019, matched with unintentional overdose decedents from the California death registry. We assessed associations between demographic and clinical characteristics with risk of overdose death, using cumulative incidence functions and Fine-Gray subdistribution hazard models. Results Of 9,860 patients residing in San Francisco, CA, USA, at the time of their trauma activation or admission during the study period, 1,418 died (4.3 per 100 person-years), 107 from unintentional overdose (0.3 per 100 person-years). Overdose decedents were 84% male, 50% white, with a mean age of 48 years at the time of presentation; 20% of deaths occurred within 3 months of hospitalization, and 40% were attributed to a prescription opioid. In multivariate analysis, younger age, male sex, white race, and having undergone a urine drug screening were all associated with subsequent death from overdose. Conclusion During a mean 3.4-year follow-up, the mortality rate from overdose among adult patients with traumatic incidents was 0.3/100 person-years. Trauma hospitalization may serve as an opportunity to screen and initiate prevention, harm reduction, and treatment interventions.

Background The onset of the COVID-19 pandemic led to the postponement of low-acuity surgical procedures in an effort to conserve resources and ensure patient safety. This study aimed to characterize patient-reported concerns about undergoing surgical procedures during the pandemic. Methods We administered a cross-sectional survey to patients who had their general and plastic surgical procedures postponed at the onset of the pandemic, asking about barriers to accessing surgical care. Questions addressed dependent care, transportation, employment and insurance status, as well as perceptions of and concerns about COVID-19. Mixed methods and inductive thematic analyses were conducted. Results One hundred thirty-five patients were interviewed. We identified the following patient concerns: contracting COVID-19 in the hospital (46%), being alone during hospitalization (40%), facing financial stressors (29%), organizing transportation (28%), experiencing changes to health insurance coverage (25%), and arranging care for dependents (18%). Nonwhite participants were 5 and 2.5 times more likely to have concerns about childcare and transportation, respectively. Perceptions of decreased hospital safety and the consequences of possible COVID-19 infection led to delay in rescheduling. Education about safety measures and communication about scheduling partially mitigated concerns about COVID-19. However, uncertainty about timeline for rescheduling and resolution of the pandemic contributed to ongoing concerns. Conclusions Providing effective surgical care during this unprecedented time requires both awareness of societal shifts impacting surgical patients and system-level change to address new barriers to care. Eliciting patients’ perspectives, adapting processes to address potential barriers, and effectively educating patients about institutional measures to minimize in-hospital transmission of COVID-19 should be integrated into surgical care.

Highlights • The COVID-19 pandemic has suspended the surgery clinical clerkship for third-year medical students at numerous institutions across the world. • In response to the pandemic, surgical educators at the University of California, San Francisco rapidly developed a virtual surgically focused curriculum to keep students connected and solidify their foundational science knowledge before their return to clinical medicine., Problem The COVID-19 pandemic has suspended the surgery clinical clerkship for third-year medical students at numerous institutions across the world. As a result, educators and students have adapted rapidly. There is a paucity of precedents regarding urgent and brusque formal curricular changes for medical students enrolled in surgical clinical rotations. Approach The University of California, San Francisco Department of Surgery created a surgically focused extended mastery learning rotation (EMLR). The surgery clerkship leadership designed a curriculum consisting of multiple learning strategies compatible with virtual learning environments. The primary aims of the newly developed EMLR were to help students consolidate their foundational science knowledge before their return to clinical medicine in an altered learning environment. The EMLR is currently underway, and further studies are necessary to evaluate its effectiveness.

Objectives To investigate the performance and perspectives of third-year medical students (MS3s) participating in near-peer learning (NPL) sessions during their core surgical clerkship following a 15-month preclerkship curriculum. Design An evaluation study of 7 NPL sessions developed and implemented by fourth-year medical students (MS4s) held from March 2019 to February 2020. MS4s taught 1-2 sessions per rotation that included test taking strategies, illness script development, radiology review, case-based multiple-choice questions, and rapid review. Participants completed a questionnaire with 11 seven-point Likert and open-ended questions after each session. Analyses included quantitative comparison of shelf score averages between NPL participants and nonparticipants and qualitative content analysis for open-ended questions. Setting Surgical clerkship at the University of California, San Francisco. Participants Forty-eight (32%) MS3s participated, with an average attendance of 10 students per rotation. Thirty-three (69%) participants completed the questionnaire. Results MS3s enjoyed the session (6.9 ± 0.4), improved their knowledge (6.8 ± 0.5), and felt more prepared for the surgery shelf examination (6.5 ± 0.6). MS4 leaders found that MS3s always wanted radiology review, and their interest in test taking strategies and illness script development declined across the clerkship year. Participants had lower shelf exam scores compared to nonparticipants (68.1 vs 71.4, respectively; p = 0.04, ES = 0.03). Shelf exam scores increased over time in both cohorts. Each group had 2 shelf exam failures. Qualitative analysis suggests that MS3s appreciated the NPL's tailored approach and exam demystification, with a desire for increased NPL integration into the clerkship. Conclusion Students participating in NPL were satisfied with the sessions. Participants may have been students who struggled as indicated by shelf exam scores and appreciated the support. The shift in preferred topics across the blocks reflects the students’ development during clerkships. Near-peer teachers should adjust sessions over time to fit students’ evolving needs.

Background: Asian Americans have the highest prevalence of hepatitis B virus (HBV) in the US. The San Francisco Hep B Free (SFHBF) campaign aimed to increase awareness and access to HBV education and services among Asian Americans in San Francisco. Purpose: We sought to examine attitudes and knowledge among Asian Americans regarding HBV at baseline (2009) and benefits of the SFHBF outreach campaign four years later (2013). Methods: Four focus groups were conducted (n=45) in 2009, followed by in-depth interviews (n=40) in 2013. Results: In 2009, many participants were misinformed about HBV symptoms and transmission. They also reported stigma associated with HBV, which hindered Asian Americans from discussing the disease and seeking services. The 2013 interviews revealed that SFHBF had contributed towards awareness of HBV screenings and vaccinations, and also instilled acute seriousness that HBV could affect them directly. Conclusion: The in-depth interviews conducted in 2013 illustrated that there was less concern about “saving face,” but a shift to a level of seriousness associated with HBV. Future efforts among Asian Americans should continue to focus on self-efficacy regarding HBV prevention, including screening and vaccination.

The CRISPR/Cas9 system facilitates precise DNA modifications by generating RNA-guided blunt-ended double-strand breaks. We demonstrate that guide RNA pairs generate deletions that are repaired with a high level of precision by non-homologous end-joining in mammalian cells. We present a method called knock-in blunt ligation for exploiting these breaks to insert exogenous PCR-generated sequences in a homology-independent manner without loss of additional nucleotides. This method is useful for making precise additions to the genome such as insertions of marker gene cassettes or functional elements, without the need for homology arms. We successfully utilized this method in human and mouse cells to insert fluorescent protein cassettes into various loci, with efficiencies up to 36% in HEK293 cells without selection. We also created versions of Cas9 fused to the FKBP12-L106P destabilization domain in an effort to improve Cas9 performance. Our in vivo blunt-end cloning method and destabilization-domain-fused Cas9 variant increase the repertoire of precision genome engineering approaches.

The ability to precisely modify the genome in a site-specific manner is extremely useful. The CRISPR/Cas9 system facilitates precise modifications by generating RNA-guided double-strand breaks. We demonstrate that guide RNA pairs generate deletions that are repaired with a high level of precision by non-homologous end-joining in mammalian cells. We present a method called knock-in blunt ligation for exploiting this excision and repair to insert exogenous sequences in a homology-independent manner without loss of additional nucleotides. We successfully utilize this method in a human immortalized cell line and induced pluripotent stem cells to insert fluorescent protein cassettes into various loci, with efficiencies up to 35.8% in HEK293 cells. We also present a version of Cas9 fused to the FKBP12-L106P destabilization domain for investigating repair dynamics of Cas9-induced double-strand breaks. Our in vivo blunt-end cloning method and destabilization-domain-fused Cas9 variant increase the repertoire of precision genome engineering approaches.