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Tina Design Suite 9 3 11

TINA is a product of DesignSoft exclusively for Texas Instruments. This complimentary version is fully functional but does not support some other features available with the full version of TINA.For a complete list of available TINA-TI models, see: SpiceRack -- A Complete List Need HSpice models to aid in your design? Our HSpice model collection can be found here.

Tina Design Suite 9 3 11

* INA129****************************************************************************** (C) Copyright 2011 Texas Instruments Incorporated. All rights reserved. ******************************************************************************* This model is designed as an aid for customers of Texas Instruments.** TI and its licensors and suppliers make no warranties, either expressed** or implied, with respect to this model, including the warranties of ** merchantability or fitness for a particular purpose. The model is** provided solely on an "as is" basis. The entire risk as to its quality** and performance is with the customer.******************************************************************************* This model is subject to change without notice. Texas Instruments* Incorporated is not responsible for updating this model.********************************************************************************* Released by: Analog eLab Design Center, Texas Instruments Inc.* Part: INA129* Date: 08JUL2011* Model Type: ALL IN ONE* Simulator: PSPICE* Simulator Version: 16.0.0.p001* EVM Order Number: N/A* EVM Users Guide: N/A* Datasheet: SBOS051B - OCTOBER 1995 - REVISED FEBRUARY 2005** Model Version: 1.0******************************************************************************* * Updates:** Version 1.0 : * Release to Web******************************************************************************* COMMENTS* CONNECTIONS: NON-INVERTING INPUT* INVERTING INPUT* POSITIVE POWER SUPPLY* NEGATIVE POWER SUPPLY* OUTPUT* REFERENCE* GAIN SENSE 1* GAIN SENSE 2* * PIN CONFIG FOR INA129 1 2 3 4 5 8 9 10 *****************************************************************************.SUBCKT INA129 1 2 3 4 5 8 9 10 X1 15 17 3 4 11 A1_129E X2 15 16 3 4 12 A2_129E X3 14 13 3 4 5 A3_129E R1 11 13 40.0000K R2 13 5 39.996K R3 12 14 40.0000K R4 14 8 40.0000K CIN 13 14 4.0000PF R1FB 9 11 24.700K CC1 17 11 5.0000PF R2FB 10 12 24.700K CC2 16 12 5.0000PF CG1 9 0 10.0000PF CG2 10 0 8.0000PF RCE 17 9 20G I1 3 16 DC 20.00E-6 I2 3 17 DC 20.00E-6 IB1CAN 3 42 DC 40.00E-9 IB2CAN 3 46 DC 40.00E-9 IBAL 0 4 DC 6.5E-6 D1 15 17 DX D2 15 16 DX Q1 16 42 10 QX Q2 17 46 9 QX V1 3 15 DC 1.700

Each of our spaces can be used separately or for that truly unique event the entire space at club café may be available for your event. Flexibility in the set-up and design of our space is limited only by your imagination.

Here we report the directed evolution of Nme2Cas9 (ref. 17), expanding its PAM scope from the N4CC requirement of the wild-type protein to include most N4YN sequences, where Y is C or T. To enable the evolution of this non-SpCas9 ortholog, we developed and integrated three technologies. First, we established a new, generalizable selection strategy requiring both PAM recognition and functional editing activity. We carried out selections in parallel across single PAM sequences using phage-assisted non-continuous evolution (PANCE)19 and a high-throughput eVOLVER-enabled20 phage-assisted continuous evolution (ePACE) platform. Last, we developed a high-throughput base editing-dependent PAM-profiling assay (BE-PPA) to rapidly and thoroughly characterize evolving Nme2Cas9 variants and to guide evolutionary trajectories. With these developments, we evolved four Nme2Cas9 variants that enable robust precision genome editing at PAMs with a single specified pyrimidine nucleotide: eNme2-C, eNme2-C.NR, eNme2-T.1 and eNme2-T.2. The evolved Nme2 variants exhibit comparable (eNme2-T.1 and eNme2-T.2) or more robust (eNme2-C) base editing and lower off-target editing than SpRY, the only other engineered variant capable of accessing similar PAMs for a subset of target sites7. Together, these new variants offer broad PAM accessibility that is complementary to the suite of PAMs previously targetable by SpCas9-derived variants. Moreover, the selection strategy developed in this study is highly scalable and general. Because of the lack of target site requirements, this selection could in principle be applied to evolve functional activities in any Cas ortholog or to optimize editing at a specific PAM or target site.

To address these limitations, we designed a new selection strategy in which the target protospacer and PAM can be fully specified without affecting the coding sequence of the gene responsible for selection survival (Fig. 1b). To achieve this programmability, we used the splicing capabilities of inteins, protein elements that insert and remove themselves from other proteins in cis, leaving only a small (roughly 3- to 10-aa) extein scar25,26. We hypothesized that trans split-inteins could function effectively as cis-splicing elements when the N- and C-inteins are fused together with a linker containing a programmed PAM and protospacer. We used the split-intein pair from N. punciforme (Npu)27 since we previously showed that gIII split after Leu 10 with the Npu intein supports robust phage propagation after trans splicing28.

Previous efforts to evolve SpCas9 on specific PAM sequences (NAG, NAC, NAT, etc.) yielded variants with both higher activity and specificity compared to variants evolved on a broad set of pooled PAMs9. Evolving on specific PAM sequences using traditional PACE methodology, however, is limited by throughput, since PACE is inherently challenging to parallelize due to cost, space and design complexity, requiring temperature-controlled rooms and fluid-handling equipment31. This constraint limits the number of conditions that can be explored in a PACE campaign, a drawback given the difficulty of predicting the set of conditions that will evolve molecules with desired properties.

Next, we developed a method to rapidly profile the PAM scope of Nme2Cas9 variants that emerge during evolution. Assessing PAM compatibility by testing individual sites in mammalian cells is throughput-limited. Although many library-based PAM-profiling methods have been described, these methods rely on nuclease activity (PAM depletion10, PAMDA7,15, TXTL PAM profiling34, CHAMP35, etc.) or Cas protein binding activity (PAM-SCANR36, CHAMP35, etc.), which may not fully reflect PAM compatibility in precision gene editing applications such as base editing. We previously reported a mammalian cell base editing profiling assay9,37; however, this method is both slower and costlier than cell-free34,35 or E. coli-based7,10,15,36 methods, making it better suited for the characterization of late-stage variants.

While the N4TN activity of eNme2-T.1 and eNme2-T.2 were promising, ABE-PPE data (Fig. 2c) suggested that these two variants may also have activity on other PAM sites. To further characterize the PAM compatibility of these variants in mammalian cells, we evaluated eNme2-T.1-ABE8e and eNme2-T.2 at 22 genomic sites flanked by N4VN PAMs (where V is A, C or G). Consistent with their evolutionary histories and with ABE-PPE showing strongest enrichment for N4TN PAMs, activity on N4VN PAM sites was generally lower than on N4TN PAM sites and varied considerably from site to site (Supplementary Fig. 18). These mammalian cell editing data suggest that while eNme2-T.1-ABE8e and eNme2-T.1-ABE8e are capable of accessing N4TN PAMs and some other PAMs, editing efficiencies especially for the latter remain site-dependent. Together, these evolved variants from both trajectories (eNme2-C, eNme2-T.1 and eNme2-T.2) offer access to a large suite of pyrimidine-rich PAMs largely inaccessible to SpCas9-derived variants.

By integrating a functional Cas enzyme selection (SAC-PACE) with high-throughput phage-assisted evolution platforms (PANCE and ePACE) and a high-throughput PAM-profiling method (BE-PPA) to guide our evolutionary campaign, we demonstrated evolution of a non-S. pyogenes Cas protein to acquire single-nucleotide PAM recognition. We developed two highly efficient, highly specific Nme2Cas9 variants capable of targeting N4CN PAM sequences across different gene editing modalities and two variants capable of adenine base editing at many N4TN PAM sequences, affording unparalleled access to pyrimidine-PAM sequences. Together, these variants complement the suite of commonly used SpCas variants and will enable the study and potential correction of previously inaccessible or poorly accessible loci, while retaining the compact size and high genome-wide specificity of Nme2Cas9 that could be beneficial to downstream clinical applications.

T.P.H. conceptualized and validated the SAC-PACE selection and the PAM-profiling assay, developed and cloned plasmids and phage, designed evolution schemes and mammalian cell experiments, conducted characterization of evolved variants in bacteria and mammalian cells and analyzed data. Z.J.H. developed and characterized the ePACE platform, conceptualized and characterized millifluidic devices and pressure regulation unit, wrote ePACE software, carried out ePACE evolution experiments, performed mutational analysis of evolved variants and analyzed data. S.M.M. performed and assisted with analysis of GUIDE-seq and validation of the ePACE platform. B.G.W. aided with conceptualization and design of millifluidic devices and assisted with validation of the ePACE platform. T.W. provided materials and assisted with analysis of the MBP evolution during ePACE validation. P.A.B. aided in fabrication and characterization of millifluidic devices. A.S.K. and D.R.L. designed and supervised the research. T.P.H., Z.J.H., A.S.K. and D.R.L. drafted the manuscript with input from all authors. 350c69d7ab


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