Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems

Microbial CRISPR-Cas systems are divided into Class 1, with multisubunit effector complexes, and Class 2, with single protein effectors. Currently, only two Class 2 effectors, Cas9 and Cpf1, are known. We describe here three distinct Class 2 CRISPR-Cas systems. The effectors of two of the identified systems, C2c1 and C2c3, contain RuvC-like endonuclease domains distantly related to Cpf1. The third system, C2c2, contains an effector with two predicted HEPN RNase domains. Whereas production of mature CRISPR RNA (crRNA) by C2c1 depends on tracrRNA, C2c2 crRNA maturation is tracrRNA independent. We found that C2c1 systems can mediate DNA interference in a 5'-PAM-dependent fashion analogous to Cpf1. However, unlike Cpf1, which is a single-RNA-guided nuclease, C2c1 depends on both crRNA and tracrRNA for DNA cleavage. Finally, comparative analysis indicates that Class 2 CRISPR-Cas systems evolved on multiple occasions through recombination of Class 1 adaptation modules with effector proteins acquired from distinct mobile elements.National Institute of Mental Health (U.S.) (Grant 5DP1-MH100706)National Institute of Mental Health (U.S.) (Grant 1R01-MH110049)National Institute of Diabetes and Digestive and Kidney Diseases (U.S.) (Grant 5R01DK097768-03)National Institutes of Health (U.S.) (Grant GM10407

Characterizing genomic alterations in cancer by complementary functional associations.

Systematic efforts to sequence the cancer genome have identified large numbers of mutations and copy number alterations in human cancers. However, elucidating the functional consequences of these variants, and their interactions to drive or maintain oncogenic states, remains a challenge in cancer research. We developed REVEALER, a computational method that identifies combinations of mutually exclusive genomic alterations correlated with functional phenotypes, such as the activation or gene dependency of oncogenic pathways or sensitivity to a drug treatment. We used REVEALER to uncover complementary genomic alterations associated with the transcriptional activation of β-catenin and NRF2, MEK-inhibitor sensitivity, and KRAS dependency. REVEALER successfully identified both known and new associations, demonstrating the power of combining functional profiles with extensive characterization of genomic alterations in cancer genomes

Acute kidney injury in patients treated with immune checkpoint inhibitors

Background: Immune checkpoint inhibitor-associated acute kidney injury (ICPi-AKI) has emerged as an important toxicity among patients with cancer. Methods: We collected data on 429 patients with ICPi-AKI and 429 control patients who received ICPis contemporaneously but who did not develop ICPi-AKI from 30 sites in 10 countries. Multivariable logistic regression was used to identify predictors of ICPi-AKI and its recovery. A multivariable Cox model was used to estimate the effect of ICPi rechallenge versus no rechallenge on survival following ICPi-AKI. Results: ICPi-AKI occurred at a median of 16 weeks (IQR 8-32) following ICPi initiation. Lower baseline estimated glomerular filtration rate, proton pump inhibitor (PPI) use, and extrarenal immune-related adverse events (irAEs) were each associated with a higher risk of ICPi-AKI. Acute tubulointerstitial nephritis was the most common lesion on kidney biopsy (125/151 biopsied patients [82.7%]). Renal recovery occurred in 276 patients (64.3%) at a median of 7 weeks (IQR 3-10) following ICPi-AKI. Treatment with corticosteroids within 14 days following ICPi-AKI diagnosis was associated with higher odds of renal recovery (adjusted OR 2.64; 95% CI 1.58 to 4.41). Among patients treated with corticosteroids, early initiation of corticosteroids (within 3 days of ICPi-AKI) was associated with a higher odds of renal recovery compared with later initiation (more than 3 days following ICPi-AKI) (adjusted OR 2.09; 95% CI 1.16 to 3.79). Of 121 patients rechallenged, 20 (16.5%) developed recurrent ICPi-AKI. There was no difference in survival among patients rechallenged versus those not rechallenged following ICPi-AKI. Conclusions: Patients who developed ICPi-AKI were more likely to have impaired renal function at baseline, use a PPI, and have extrarenal irAEs. Two-thirds of patients had renal recovery following ICPi-AKI. Treatment with corticosteroids was associated with improved renal recovery

Acute kidney injury in patients treated with immune checkpoint inhibitors

BACKGROUND: Immune checkpoint inhibitor-associated acute kidney injury (ICPi-AKI) has emerged as an important toxicity among patients with cancer. METHODS: We collected data on 429 patients with ICPi-AKI and 429 control patients who received ICPis contemporaneously but who did not develop ICPi-AKI from 30 sites in 10 countries. Multivariable logistic regression was used to identify predictors of ICPi-AKI and its recovery. A multivariable Cox model was used to estimate the effect of ICPi rechallenge versus no rechallenge on survival following ICPi-AKI. RESULTS: ICPi-AKI occurred at a median of 16 weeks (IQR 8-32) following ICPi initiation. Lower baseline estimated glomerular filtration rate, proton pump inhibitor (PPI) use, and extrarenal immune-related adverse events (irAEs) were each associated with a higher risk of ICPi-AKI. Acute tubulointerstitial nephritis was the most common lesion on kidney biopsy (125/151 biopsied patients [82.7%]). Renal recovery occurred in 276 patients (64.3%) at a median of 7 weeks (IQR 3-10) following ICPi-AKI. Treatment with corticosteroids within 14 days following ICPi-AKI diagnosis was associated with higher odds of renal recovery (adjusted OR 2.64; 95% CI 1.58 to 4.41). Among patients treated with corticosteroids, early initiation of corticosteroids (within 3 days of ICPi-AKI) was associated with a higher odds of renal recovery compared with later initiation (more than 3 days following ICPi-AKI) (adjusted OR 2.09; 95% CI 1.16 to 3.79). Of 121 patients rechallenged, 20 (16.5%) developed recurrent ICPi-AKI. There was no difference in survival among patients rechallenged versus those not rechallenged following ICPi-AKI. CONCLUSIONS: Patients who developed ICPi-AKI were more likely to have impaired renal function at baseline, use a PPI, and have extrarenal irAEs. Two-thirds of patients had renal recovery following ICPi-AKI. Treatment with corticosteroids was associated with improved renal recovery

Diversity and evolution of class 2 CRISPR–Cas systems

Class 2 CRISPR-Cas systems are characterized by effector modules that consist of a single multidomain protein, such as Cas9 or Cpf1. We designed a computational pipeline for the discovery of novel class 2 variants and used it to identify six new CRISPR-Cas subtypes. The diverse properties of these new systems provide potential for the development of versatile tools for genome editing and regulation. In this Analysis article, we present a comprehensive census of class 2 types and class 2 subtypes in complete and draft bacterial and archaeal genomes, outline evolutionary scenarios for the independent origin of different class 2 CRISPR-Cas systems from mobile genetic elements, and propose an amended classification and nomenclature of CRISPR-Cas. Keywords: Bacterial evolution; Bacterial genetics; CRISPR-Cas systemsUS National Institute of Mental Health (Grant 5DP1-MH100706)US National Institute of Mental Health (Grant 1R01-MH110049