Altering, Improving, And Defining The Specificities Of Crispr-Cas Nucleases

CRISPR-Cas9 nucleases have been widely adopted for genome editing applications to knockout genes or to introduce desired changes. While these nucleases have shown immense promise, two notable limitations of the wild-type form of the broadly used Streptococcus pyogenes Cas9 (SpCas9) are the restriction of targeting range to sites that contain an NGG protospacer adjacent motif (PAM), and the undesirable ability of the enzyme to cleave off-target sites that resemble the on-target site. Scarcity of PAM motifs can limit implementations that require precise targeting, whereas off-target effects can confound research applications and are important considerations for therapeutics. To improve the targeting range of SpCas9 and an orthogonal Cas9 from Staphylococcus aureus (called SaCas9), we optimized a heterologous genetic selection system that enabled us to perform directed evolution of PAM specificity. With SpCas9, we evolved two separate variants that can target NGA and NGCG PAMs1, and with SaCas9 relaxed the PAM from NNGRRT to NNNRRT2, increasing the targetability of these enzyme 2- to 4-fold. The genome-wide specificity profiles of SpCas9 and SaCas9 variants, determine by GUIDE-seq3, indicate that they are at least as, if not more, specific than the wild-type enzyme1,2. Together, these results demonstrate that the inherent PAM specificity of multiple different Cas9 orthologues can be purposefully modified to improve the accuracy of targeting. Existing strategies for improving the genome-wide specificity of SpCas9 have thus far proven to be incompletely effective and/or have other limitations that constrain their use. To address the off-target potential of SpCas9, we engineered a high-fidelity variant of SpCas9 (called SpCas9-HF1), that contains alterations designed to reduce non-specific contacts to the target strand DNA backbone. In comparison to wild-type SpCas9, SpCas9-HF1 rendered all or nearly all off-target events imperceptible by GUIDE-seq and targeted deep-sequencing methods with standard non-repetitive target sites in human cells4. Even for atypical, repetitive target sites, the vast majority of off-targets induced by SpCas9-HF1 and optimized derivatives were not detected4. 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. Finally, on-target activity and genome-wide specificity are two important properties of engineered nucleases that should be characterized prior to adoption of such technologies for research or therapeutic applications. CRISPR-Cas Cpf1 nucleases have recently been described as an alternative genome-editing platform5, yet their activities and genome-wide specificities remain largely undefined. Based on assessment of on-target activity across more than 40 target sites, we demonstrate that two Cpf1 orthologues function robustly in human cells with efficiencies comparable to those of the widely used Streptococcus pyogenes Cas9. We also demonstrate that four to six bases at the 3’ end of the short CRISPR RNA (crRNA) used to program Cpf1 are insensitive to single base mismatches, but that many of the other bases within the crRNA targeting region are highly sensitive to single or double substitutions6. Consistent with these results, GUIDE-seq performed in multiple cell types and targeted deep sequencing analyses of two Cpf1 nucleases revealed no detectable off-target cleavage for over half of 20 different crRNAs we examined. Our results suggest that the two Cpf1 nucleases we characterized generally possess robust on-target activity and high specificities in human cells, findings that should encourage broader use of these genome editing enzymes. 1. Kleinstiver, BP, et al. (2015) Nature, 523(7561):481-5 2. Kleinstiver, BP, et al. (2015) Nature Biotechnology, 33(12):1293-98 3. Tsai, SQ et al. (2015) Nature Biotechnology, 33(2):187-97 4. Kleinstiver, BP and Pattanayak, V, et al. (2016), Nature, 529(7587):490-5 5. Zetsche, B, et al. (2015) Cell, 163(3):759-71 6. Kleinstiver, BP and Tsai, SQ, et al. (2016), Nature Biotechnology, 34(8):869-7

The mechanisms associated with reflux episodes in ambulant subjects with gastro-esophageal reflux disease

Background: The mechanisms associated with gastro-esophageal reflux (GER) episodes were studied using combined High-resolution Impedance Manometry (HRIM) and pH monitoring in ambulant subjects with different patterns of GERD. Methods: Sixteen subjects with mild-moderate esophagitis (Los Angeles (LA) grade A&B) (group A) and 11 subjects with severe esophagitis (LA grade C&D) or Barrett's esophagus (BE) were studied before and after a meal, resting, while walking, and during standardized exercise, using a HRIM and a pH probe. Key Results: Post-prandial acid GER episodes were more common in group B (median 10 range (3-18) vs A (6.5 (0-18), p = 0.048). Postprandial acid clearance time was much longer in group B (median 0.71(0.07-2.66 min) vs A (0.17 (0.04-2.44 min), p = 0.02). Transient lower esophageal sphincter relaxation (TLESR) was the most frequent mechanism associated with GER episodes in both groups. Post-prandial TLESRs with GER were more common in group B (median 17 (9-24) vs A 13.5 (7-34), p = 0.014), particularly during exercise (B 8 (6-9) vs A 6 (5-6.8), p = 0.007). Post-prandially TLESR with acid reflux increased during exercise in both groups (A rest median 2.4 (0-6.4) per hour vs exercise 4.7 (0-17.3), p = 0.005 and B 4 (0.8-9.6) vs 5.3 (2.7-13.3) per hour, p = 0.045). Conclusions and Inferences: TLESR was the most common mechanism associated with reflux episodes in all subjects. Acid reflux episodes were more common in subjects with severe esophagitis or BE and esophageal acid clearance was much slower. Post-prandial exercise increased TLESR with acid reflux and GERD patients should be encouraged to avoid exercise immediately after a meal

High-fidelity CRISPR-Cas9 variants with undetectable genome-wide off-targets

CRISPR-Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. Existing strategies for reducing genome-wide off-targets of the broadly 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 harboring 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. Strikingly, 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-targets induced by SpCas9-HF1 were not detected. 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 RNA-guided nucleases

Precision Targeting of BFL-1/A1 and an ATM Co-dependency in Human Cancer

Summary: Cancer cells overexpress a diversity of anti-apoptotic BCL-2 family proteins, such as BCL-2, MCL-1, and BFL-1/A1, to enforce cellular immortality. Thus, intensive drug development efforts have focused on targeting this class of oncogenic proteins to overcome treatment resistance. Whereas a selective BCL-2 inhibitor has been FDA approved and several small molecule inhibitors of MCL-1 have recently entered phase I clinical testing, BFL-1/A1 remains undrugged. Here, we developed a series of stapled peptide design principles to engineer a functionally selective and cell-permeable BFL-1/A1 inhibitor that is specifically cytotoxic to BFL-1/A1-dependent human cancer cells. Because cancers harbor a diversity of resistance mechanisms and typically require multi-agent treatment, we further investigated BFL-1/A1 co-dependencies by mining a genome-scale CRISPR-Cas9 screen. We identified ataxia-telangiectasia-mutated (ATM) kinase as a BFL-1/A1 co-dependency in acute myeloid leukemia (AML), which informed the validation of BFL-1/A1 and ATM inhibitor co-treatment as a synergistic approach to subverting apoptotic resistance in cancer. : Guerra et al. constructed an exquisitely selective BFL-1 inhibitor capable of covalent BFL-1 targeting and cellular penetrance without membrane disruption. Mining a genetic dependency database revealed a spectrum of BFL-1 dependency in cancer and an ATM co-dependency in AML, prompting the combination of BFL-1 and ATM inhibitors to achieve synergistic cytotoxicity. Keywords: BFL-1, A1, BCL-2 family, apoptosis, stapled peptide, covalent inhibitor, dependency, ATM, AML, cance