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CRISPR-Cas9 nuclease-based gene drives have been developed toward the aim of control of the human malaria vector Anopheles gambiae . Gene drives are based on an active source of Cas9 nuclease in the germline that promotes super-Mendelian inheritance of the transgene by homology-directed repair (“homing”). Understanding whether CRISPR-induced off-target mutations are generated in Anopheles mosquitoes is an important aspect of risk assessment before any potential field release of this technology. We compared the frequencies and the propensity of off-target events to occur in four different gene-drive strains, including a deliberately promiscuous set-up, using a nongermline restricted promoter for SpCas9 and a guide RNA with many closely related sites (two or more mismatches) across the mosquito genome. Under this scenario we observed off-target mutations at frequencies no greater than 1.42%. We witnessed no evidence that CRISPR-induced off-target mutations were able to accumulate (or drive) in a mosquito population, despite multiple generations’ exposure to the CRISPR-Cas9 nuclease construct. Furthermore, judicious design of the guide RNA used for homing of the CRISPR construct, combined with tight temporal constriction of Cas9 expression to the germline, rendered off-target mutations undetectable. The findings of this study represent an important milestone for the understanding and managing of CRISPR-Cas9 specificity in mosquitoes, and demonstrates that CRISPR off-target editing in the context of a mosquito gene drive can be reduced to minimal levels.

Keratinocyte carcinoma (KC), defined as squamous cell carcinoma and basal cell carcinoma, is the most common malignancy among white, non-Hispanic renal transplant recipients. Although recent genome-wide association studies reported that class II HLA is associated with KC risk, epidemiologic data on HLA type and KC risk in renal transplant recipients is limited. Using an institutional cohort of white, non-Hispanic renal transplant recipients transplanted between 1993 and 2017, we examined the association between pretransplant molecular HLA types and KC risk. Posttransplant KCs were captured using the International Classification of Diseases codes and validated using pathology reports. Cox proportional hazards regression models were used to estimate hazard ratios of incident KC, squamous cell carcinoma, and basal cell carcinoma, adjusting for age, male sex, history of KC, Charlson comorbidity index, HLA mismatch, transplant type, year of transplant, and the type of immunosuppression. Among 617 subjects (mean age 53 years, 67% male), 10% developed posttransplant KC. Multivariable Cox regression analyses showed HLA-DRB1∗13 was associated with KC risk (hazard ratio, 1.84; 95% confidence interval, 1.00–3.38) and squamous cell carcinoma risk (hazard ratio, 2.24; 95% confidence interval, 1.12–4.49), whereas HLA-DRB1∗14 (hazard ratio, 2.81; 95% confidence interval, 1.14–6.91) was associated with basal cell carcinoma risk. Our findings suggest that a subset of renal transplant recipients with specific HLA polymorphisms may be at increased KC risk.

Schaefer et al. recently advanced the provocative conclusion that CRISPR-Cas9 nuclease can induce off-target alterations at genomic loci that do not resemble the intended on-target site. Using high-coverage whole genome sequencing (WGS), these authors reported finding SNPs and indels in two CRISPR-Cas9-treated mice that were not present in a single untreated control mouse. On the basis of this association, Schaefer et al. concluded that these sequence variants were caused by CRISPR-Cas9. This new proposed CRISPR-Cas9 off-target activity runs contrary to previously published work and, if the authors are correct, could have profound implications for research and therapeutic applications. Here we demonstrate that the simplest interpretation of Schaefer et al.9s data is that the two CRISPR-Cas9-treated mice are actually more closely related genetically to each other than to the control mouse. This strongly suggests that the so-called “unexpected mutations” simply represent SNPs and indels shared in common by these mice prior to nuclease treatment. In addition, given the genomic and sequence distribution profiles of these variants, we show that it is challenging to explain how CRISPR-Cas9 might be expected to induce such changes. Finally, we argue that the lack of appropriate controls in Schaefer et al.9s experimental design precludes assignment of causality to CRISPR-Cas9. Given these substantial issues, we urge Schaefer et al. to revise or re-state the original conclusions of their published work so as to avoid leaving misleading and unsupported statements to persist in the literature.

CRISPR-Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. Existing strategies for reducing genome-wide off-target effects of the widely used Streptococcus pyogenes Cas9 (SpCas9) are imperfect, possessing only partial or unproven efficacies and other limitations that constrain their use. Here we describe SpCas9-HF1, a high-fidelity variant harbouring alterations designed to reduce non-specific DNA contacts. SpCas9-HF1 retains on-target activities comparable to wild-type SpCas9 with >85% of single-guide RNAs (sgRNAs) tested in human cells. Notably, with sgRNAs targeted to standard non-repetitive sequences, SpCas9-HF1 rendered all or nearly all off-target events undetectable by genome-wide break capture and targeted sequencing methods. Even for atypical, repetitive target sites, the vast majority of off-target mutations induced by wild-type SpCas9 were not detected with SpCas9-HF1. With its exceptional precision, SpCas9-HF1 provides an alternative to wild-type SpCas9 for research and therapeutic applications. More broadly, our results suggest a general strategy for optimizing genome-wide specificities of other CRISPR-RNA-guided nucleases.

Schaefer et al. recently advanced the provocative conclusion that CRISPR-Cas9 nuclease can induce off-target alterations at genomic loci that do not resemble the intended on-target site.1 Using high-coverage whole genome sequencing (WGS), these authors reported finding SNPs and indels in two CRISPR-Cas9-treated mice that were not present in a single untreated control mouse. On the basis of this association, Schaefer et al. concluded that these sequence variants were caused by CRISPR-Cas9. This new proposed CRISPR-Cas9 off-target activity runs contrary to previously published work2–8 and, if the authors are correct, could have profound implications for research and therapeutic applications. Here, we demonstrate that the simplest interpretation of Schaefer et al.’s data is that the two CRISPR-Cas9-treated mice are actually more closely related genetically to each other than to the control mouse. This strongly suggests that the so-called “unexpected mutations” simply represent SNPs and indels shared in common by these mice prior to nuclease treatment. In addition, given the genomic and sequence distribution profiles of these variants, we show that it is challenging to explain how CRISPR-Cas9 might be expected to induce such changes. Finally, we argue that the lack of appropriate controls in Schaefer et al.’s experimental design precludes assignment of causality to CRISPR-Cas9. Given these substantial issues, we urge Schaefer et al. to revise or re-state the original conclusions of their published work so as to avoid leaving misleading and unsupported statements to persist in the literature.

Directly modulating the activity of genome-editing proteins has the potential to increase their specificity by reducing activity following target locus modification. We developed Cas9 nucleases that are activated by the presence of a cell-permeable small molecule by inserting an evolved 4-hydroxytamoxifen (4-HT)-responsive intein at specific positions in Cas9. In human cells, conditionally active Cas9s modify target genomic sites with up to 25-fold higher specificity than wild-type Cas9.

The RNA-programmable Cas9 endonuclease cleaves double-stranded DNA at sites complementary to a 20-base-pair guide RNA. The Cas9 system has been used to modify genomes in multiple cells and organisms, demonstrating its potential as a facile genome-engineering tool. We used in vitro selection and high-throughput sequencing to determine the propensity of eight guide-RNA:Cas9 complexes to cleave each of 10(12) potential off-target DNA sequences. The selection results predicted five off-target sites in the human genome that were confirmed to undergo genome cleavage in HEK293T cells upon expression of one of two guide-RNA:Cas9 complexes. In contrast to previous models, our results show that guide-RNA:Cas9 specificity extends past a 7- to 12-base-pair seed sequence. Our results also suggest a tradeoff between activity and specificity both in vitro and in cells as a shorter, less-active guide RNA is more specific than a longer, more-active guide RNA. High concentrations of guide-RNA:Cas9 complexes can cleave off-target sites containing mutations near or within the PAM that are not cleaved when enzyme concentrations are limiting.

Engineered zinc-finger nucleases (ZFNs) are promising tools for genome manipulation, and determining off-target cleavage sites of these enzymes is of great interest. We developed an in vitro selection method that interrogates 10(11) DNA sequences for cleavage by active, dimeric ZFNs. The method revealed hundreds of thousands of DNA sequences, some present in the human genome, that can be cleaved in vitro by two ZFNs: CCR5-224 and VF2468, which target the endogenous human CCR5 and VEGFA genes, respectively. Analysis of identified sites in one cultured human cell line revealed CCR5-224-induced changes at nine off-target loci, though this remains to be tested in other relevant cell types. Similarly, we observed 31 off-target sites cleaved by VF2468 in cultured human cells. Our findings establish an energy compensation model of ZFN specificity in which excess binding energy contributes to off-target ZFN cleavage and suggest strategies for the improvement of future ZFN design.

Background Anaplasmosis often presents with fever and headache, and typical laboratory abnormalities include leukopenia and thrombocytopenia. Polymerase chain reaction (PCR) is the preferred diagnostic method. At our institution, Anaplasma PCR is overutilized, with a low positivity rate. To improve utilization, we performed a two-part study. Part 1 determined if complete blood count (CBC) values could exclude Anaplasma infection. A stewardship algorithm was implemented during part 2 of the study via a mock stewardship protocol. Methods Part 1: Anaplasma PCR tests were included over a 3-year period. For each PCR result, white blood cell (WBC) and platelet (PLT) results from a sample collected near the time of PCR were evaluated in R Studio and GraphPad Prism. The significance of differences between PCR-positive and -negative cases was determined using the Mann–Whitney test. Cut-off values were chosen to maximize sensitivity of a screening algorithm for the detection of Anaplasma infection. Part 2: Mock stewardship was performed for 6 months. Screening criteria generated in phase 1 were applied to determine whether Anaplasma PCR would have been approved or rejected if stewardship were implemented based on laboratory and clinical parameters. Results Part 1: 2166 PCR tests were included. Patients with a positive Anaplasma PCR had lower median WBC and PLT counts than those with a negative result (Figure 1). Combining criteria of a WBC ≥11 K/µL and PLT ≥300 K/µL provided rejection criteria with 100% sensitivity and 25% specificity after excluding immunocompromised or unstable patients (Figure 2). Part 2: 663 PCR tests were analyzed. Of those, 155 (23%) met CBC rejection criteria and were reviewed by committee. The committee mock refused 110 (71%) tests and mock accepted 49 (29%) based on clinical criteria. Of the patients with positive Anaplasma PCR, 1 met CBC rejection criteria and was mock refused by committee. On review, the patient was completing treatment for Anaplasmosis, indicating limited utility of testing. None of the 45 samples that were mock accepted by clinical criteria was positive by PCR, demonstrating no additional benefit of chart review. Conclusion Implementation of a CBC-based stewardship algorithm would reduce unnecessary Anaplasma PCR testing and decrease cost by 23%. Disclosures J. Baron, Roche Diagnostics: Possible Consulting Role—not finalized at this point, No compensation yet—compensation TBD

CRISPR-Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations at genomic locations that resemble the intended target. These so-called off-target effects can confound research applications and are important considerations for potential therapeutic use. Existing strategies for reducing genome-wide off-targets of the broadly used Streptococcus pyogenes Cas9 (SpCas9) have thus far proven to be imperfect by possessing only partial efficacy and/or other limitations that constrain their use. Here we describe a high-fidelity variant of SpCas9, called SpCas9-HF1, that contains alterations in the amino acid sequence designed to reduce non-specific contacts to the target strand DNA. SpCas9-HF1 retains on-target activities comparable to wild-type SpCas9 with >85% of the 37 single-guide RNAs (sgRNAs) tested in human cells. Strikingly, with eight different sgRNAs targeted to standard non-repetitive sequences in human cells, SpCas9-HF1 rendered all or nearly all off-target events imperceptible by genome-wide break capture and targeted sequencing methods. Even for atypical, repetitive target sites, the vast majority of off-targets induced by SpCas9-HF1 were not detected. With its exceptional precision, SpCas9-HF1 provides an important and easily employed alternative to wild-type SpCas9 that can eliminate off-target effects when using CRISPR-Cas9 for research and therapeutic applications. Our findings also suggest a general strategy for improving or optimizing the genome-wide specificities of other Cas9 orthologues and engineered variants.

Gene-editing nucleases enable targeted modification of DNA sequences in living cells, thereby facilitating efficient knockout and precise editing of endogenous loci. Engineered nucleases also have the potential to introduce mutations at off-target sites of action. Such unintended alterations can confound interpretation of experiments and can have implications for development of therapeutic applications. Recently, two improved methods for identifying the off-target effects of zinc finger nucleases (ZFNs) were described–one using an in vitro cleavage site selection method and the other exploiting the insertion of integration-defective lentiviruses into nuclease-induced double-stranded DNA breaks. However, application of these two methods to a ZFN pair targeted to the human CCR5 gene led to identification of largely non-overlapping off-target sites, raising the possibility that additional off-target sites might exist. Here, we show that in silico abstraction of ZFN cleavage profiles obtained from in vitro cleavage site selections can greatly enhance the ability to identify potential off-target sites in human cells. Our improved method should enable more comprehensive profiling of ZFN specificities.