Methods and compositions for single-cell functional genomics

Abstract

Aspects of the technology relate to methods and compositions for multiplexed assessment of perturbations with single cell molecular resolution using a regulatable Cas enzyme with intrinsic RNase activity.

Description

[0001] METHODS AND COMPOSITIONS FOR SINGLE-CELL FUNCTIONAL GENOMICS BACKGROUND

[0002] Single-cell CRISPR (Perturb-seq) screens combine CRISPR-based gene perturbations with single-cell RNA sequencing (scRNA-seq) to analyze gene function and regulatory networks at single-cell resolution. By introducing a pooled sgRNA library into cells, each carrying a single perturbation, and profiling the resulting transcriptomic changes, Perturb-seq enables the simultaneous study of numerous genetic perturbations within heterogeneous populations. This approach provides insights into gene interactions, cellular state transitions, and context-specific responses to perturbations Perturb-seq has broad applications, including identifying gene functions, mapping regulatory networks, uncovering disease mechanisms, and discovering therapeutic targets.

[0003] SUMMARY

[0004] Single-cell CRISPR (Perturb-seq) screens have primarily relied on Cas9 for inducing loss-of-function phenotypes, whereas Cas 12a, despite its unique effectiveness for multiplex guide expression, remains underexplored. This may be due to Cas 12a's guide RNA array (pre-crRNA) self-processing activity and the subsequent challenges associated with pre- crRNA sequence recovery during single-cell RNA sequencing library’ preparation. By developing modified pre-crRNA expression vectors and a degron-based, enhanced Cas 12a system for gene knock-out, the self-processing constraint was overcome. Described herein are methods and compositions applicable to across cell types, target genes, and with minimized guide RNA library’ needs, that allow for accurate detection of pre-crRNAs and gene editing-induced effects on the transcriptome at the single-cell level. Some aspects of the technology relate to compositions and methods comprising improved Casl2a-transcription repressor fusion proteins for individual or multi-gene suppression. The methods and compositions described herein thus greatly expand the possibilities for future Perturb-seq efforts and broader application of Cas 12a for genetic disruption at-scale.

[0005] Some aspects of the technology relate to a method for multiplexed assessment of perturbations with single cell molecular resolution, the method comprising: (a) delivering a vector encoding a pre-crRNA array comprising a barcode and multiple crRNAs to an initial population of cells, w'herein cells of the initial population comprise a repressible Cas 12a enzyme; (b) culturing the initial population of cells to effect Casl2a enzyme -mediated perturbation, thereby producing a population of perturbed cells: (c) repressing pre-crRNA processing activity of the repressible Casl2a enzyme; and (d) analyzing RNA sequences (e.g., mRNA sequences) of individual perturbed cells of the population of perturbed cells using a single-cell gene expression technique.

[0006] In some embodiments, individual ceils of the initial population of cells receive at least one perturbation, and the barcode of the pre-crRNA array comprises a sequence identifying the perturbation.

[0007] In some embodiments, the perturbations are selected from genetic perturbations and epigenetic perturbations. In some embodiments, the genetic perturbations are selected from deletions, insertions, and substitutions. In some embodiments, the epigenetic perturbations are selected from methylation, acetylation, and phosphorylation.

[0008] In some embodiments, the multiple crRNAs target a single gene of interest. In some embodiments, the multiple crRNAs target at least two different regions of the single gene of interest. In some embodiments, multiple crRNAs target two or more genes of interest.

[0009] In some embodiments, each pre-crRNA of the pre-crRNA array comprises, in the following order, a direct repeat (DR) domain, a Spacer domain, and a 3' end

[0010] In some embodiments, each pre-crRNA of the pre-crRNA array further comprises a capture sequence. In some embodiments, the capture sequence is at the 3' end of each pre- crRNA.

[0011] In some embodiments, step (a) of the method comprises delivering a pooled library of vectors, each encoding a pre-crRNA array comprising multiple crRN As to the initial population of ceils.

[0012] In some embodiments, the repressible Casl2a enzyme comprises a degron domain. In some embodiments, the degron domain is a C-terminal FKBP12F36Vfission domain, and (c) comprises culturing the initial population of cells in the presence of a dTag small molecule dTagVl.

[0013] In some embodiments, the repressible Casl 2a enzyme is a repressible enhanced Acidaminococcus sp. Casl2a (enAs)Cas!2a enzyme, optionally wherein the repressible (enAs)Casl 2a enzyme comprises the amino acid sequence of SEQ ID NO: 1,

[0014] In some embodiments, the repressible Casl2a enzyme is a repressible deactivated Cas 12a (dCas 12a) enz me.

[0015] In some embodiments, the repressible dCasl2a enzyme is linked to a transcription repressor domain, optionally selected from KRAB (Kriippel-associated box), SID (Sin3 Interaction Domain), MeCP2 (Methyl-CpG-Binding Protein 2), LSD1 (Lysine-Specific Demethylase 1), dCas9-Mxil, CoREST, Ezh2 (Enhancer of Zeste Homolog 2), HP1 (Heterochromatin Protein 1), REST (Repressor Element- 1 Silencing Transcription Factor), Polycomb Repressor Complex (PRC2), ZIM3 (Zinc Finger Imprinted 3), DNMT3A (DNA methyltransferase 3 alpha), and DNMT3L (DMA methyltransferase 3 like).

[0016] In some embodiments, the repressible dCas!2a enzyme is linked to a transcription activation domain, optionally selected from VP64, p65, Rta (Replication and Transcription Activator), VPR (VP64-p65-Rta), SunTag, HSF1 (Heat Shock Factor 1), P300 Core, SAM (Synergistic Activation Mediator), and ET1 (Epstein-Barr Virus EBNA-LP Transactivation Domain).

[0017] In some embodiments, the vector is a non-viral vector In some embodiments, the non-viral vector is selected from plasmid DNA, minicircle DNA, transposons, and artificial chromosomes.

[0018] In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is selected from lentiviral vectors, adenoviral vectors, adeno-associated viral (AAV) vectors, retroviral vectors, herpes simplex virus (HSV) vectors, sendai virus (SeV) vectors, and baculovirus vectors.

[0019] In some embodiments, delivering of the vector compri ses transfecting or transducing cells of the population with the vector such that single cells of the initial population receive a single pre-crRNA array.

[0020] In some embodiments, the single-cell gene expression technique comprises single-cell RNA sequencing.

[0021] In some embodiments, the method further comprises determining measured differences in the RN A sequences (e.g., mRNA sequences) that are correlated to perturbations detected for individual perturbed cells.

[0022] In some embodiments, the population of cells comprises primary cells and / or immortalized cells.

[0023] Other aspects of the technology relate to a method for multiplexed assessment of perturbations with single cell molecular resolution, the method comprising: (a) culturing an initial population of cells comprising (i) a pre-crRNA array comprising a barcode and multiple crRNAs and (ii) a repressible Casl2a enzyme to effect Casl2a enzyme-mediated perturbation, thereby producing a population of perturbed cells; and (b) repressing pre- crRNA processing activity' of the repressible Cas12a enzyme In some embodiments, the method further comprises analyzing RNA sequences (e.g., mRNA sequences) of individual perturbed cells of the population of perturbed ceils using a single-cell gene expression technique.

[0024] Some aspects of the technology relate to a Casl2a enzyme linked to a repressible domain.

[0025] In some embodiments, the repressible domain comprises a degron domain.

[0026] In some embodiments, the Casl2a enzy me comprises an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, tire Casl2a enzyme comprises an amino acid sequence having a at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 1.

[0027] Other aspects of the technology relate to a composition comprising a vector encoding a repressible Casl2a enzyme and a vector encoding a pre-crRNA array.

[0028] In some embodiments, the composition comprises multiple vectors, each encoding a pre-crRNA array.

[0029] Yet other aspects of the technology relate to a cell comprising a vector encoding a pre-crRNA array, wherein the cell expresses a repressible Cas12a enzyme.

[0030] Hie Summary above is non-limiting.

[0031] BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figs. 1A-1G show Casl2a impairs guide detection in scRNA-seq. Fig. 1A depicts the experimental strategy for C las 12a CRISPR screen with scRNA-seq read-out. Fig. IB shows the epigenetic factors selected for the screen. Factors were selected by gene expression in H358 cells and diversity of function. Libraries contained pre-crRN As with two guides for each factor. Fig. 1 C shows the pre-crRNA construct in CROP-seq format. Fig, ID is the protocol for amplifying pre-crRNA sequences from single-cell cDNA showing representative analytical gel electrophoresis. Fig. IE shows the signal from representative pre-crRNA obtained by full-length and barcode detection methods. Fig. IF shows the percentage of cells identified as expressing none, single, or multiple pre-crRNA constructs. Fig. 1G shows the percentage of cells with consistent pre-crRNA identify calls between full-length and barcode methods.

[0033] Figs, 2A-2H show degradation of Casl2a restores pre-crRNA detection. Fig. 2A depicts the experimental strategy. Fig. 2B shows the quantification of FACS data comparing Cas12a-FKBP12F36Vand Casl2a-mediated editing efficiency and prevention of editing in the presence of dTagvl. Bar heights represent the Mean of three biological replicates normalized by average NTC values. Error bars represent SD. Statistical comparison was performed by an unpaired two-sample t-test with Welch’s correction whereby *p < 0.05, * *p < 0.01, ***p < 0.001, ****p < 0.0001 as compared to the NTC. Fig. 2C depicts the experimental strategy for scRNA-seq with Cas12a-FKBP12F36Vand incorporation of dTagVl addition to build-up pre-crRNA post gene targeting. Fig. 2D shows the UMI signal from representative pre-crRNA obtained from barcodes after 24 hours or 48 hours dTagVl treatment. FIG, 2D is a boxplot of UMI signals for all pre-crRNA in the library' obtained from barcodes after 24 hours or 48 hours dTagV 1 treatment. Statistical comparison between samples was performed by a Mann-Whitney U test whereby the p-values were 0 (Casl2a), 3.07e‘279(24 hours), and 1.32e‘61(48 hours), as compared to the first sample (no Casl2a). Fig. 2F shows the percentage of cells identified as expressing none, a single or multiple pre-crRNAs based on the barcode sequence from the epigenetic factor screen described in Fig. 2C. Figs. 2G-2H are volcano plots of transcriptome-wide changes in cells with XDAf / rf or NA TJ ^-targeting pre-cRNA vs NTCs. Genes highlighted in red have been reported in tire literature. Fig. 21 shows the top 5 biological pathways from gene ontology analysis of significantly altered genes shown in h with red asterisks highlighting the pathways reported to be affected by NAT10 perturbation before. Black bars represent fold enrichment values, while grey bars show -logic FDR-adjusted p-values.

[0034] Figs. 3A-3E show the capture Sequences at Casl2a guide's 3' end do not impair guide efficiency. Fig. 3A is a schematic of pre-crRNA expressed from direct-capture Perturb-seq formats. Fig. 3B shows the design of pre-crRNA with capture sequences added at three different positions. Fig. 3C depicts the experimental procedure. Fig. 3D is an example of FACS cell gating strategy to record mean fluorescence intensity (MFI) and % of positive cells for a particular channel. Fig, 3E shows the quantification of FACS data. Only capture sequences (CS) at the 3' end of the Casl2a guide allows for efficient editing. NTC = non¬ targeting control. Bar heights represent the Mean of two biological replicates normalized by¬ average NTC values. Error bars represent SD. Statistical comparison between guide formats was performed by an unpaired two-sample t-test with Welch’s correction whereby *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 as compared to the NTC (Guide #25).

[0035] Figs. 4A-4D show Casl2a performs efficient multiplex targeting with pre-crRNA from different construct formats. Fig 4A are schematics of pre-crRNA expressing constructs and design of pre-crRNA array with dual and quad guides found within both formats. In CROP-seq pre-crRNA is part of the 3 ’ LTR, which gets duplicated during lenti viral genome integration. This creates a second copy of pre-crRN A, which produces functional Pol III transcripts. pre-crRNA that is part of long Pol II puroRtranscript is the version detected in scRNA-seq from its polyA. The U6 promoter downstream of puroRhas been shown not to produce RNA. However, unlike Cas9, Casl2a can probably process crRNAs out of the long Pol II transcript, which would increase the amount of available guide RNA. Fig. 4B shows the quantification of FACS data obtained from comparing dual and quad pre-crRNA arrays in CROP-seq and direct-capture Perturb-seq (CapPERTURB-seq) formats. NTC = non-targeting control. Bar heights represent the Mean of three biological replicates normalized by average NIC values. Error bars represent SD. Statistical comparison between guide formats was performed by an unpaired two-sample t-test with Welch’s correction whereby *p < 0.05, **p <0.01, ***p< 0.001, ****p< 0.0001 as compared to the NTC (Guides #25, 109, 111, 113). Fig. 4C depicts the design of the last position in a pre-crRNA array expressing a sub- optimal CD81 guide followed by a capture sequence. Fig. 4D shows the quantification of FACS data obtained 'ith the pre-crRNA set-up from Fig. 4C, Bar heights represent the Mean of t 'o biological replicates normalized by average NTC values. Error bars represent SD. Statistical comparison between guide formats was performed by an unpaired two-sample t-test with Welch’s correction whereby *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 as compared to the NTC (Guide #25),

[0036] Fig. 5 shows the bioinformatic workflow for identification of pre-crRNA. Schematic of workflow used to identify pre-crRNA whereby reads (R2) from FASTQ files from a library of amplified full-length pre-crRNA are trimmed (if necessary’) and aligned to a custom reference genome using 1 Ox Genomics Cell Ranger v7.1.0. Custom reference genome is also generated by Cell Ranger based on pre-crRNA sequences in the library. Post reads alignment, the raw feature barcode matrix file is filtered to extract pre-crRNA counts by cell barcode allowing for pre-crRNA status assignment to individual cells.

[0037] Figs. 6A-6I show pre-crRNA format optimization. Fig. 6A is a scheme showing Direct-capture Perturb-seq construct and protocol for generation of the CRISPR guide libraries, when short capture sequences attached to guide RNA binds to complementary capture sequences part of oligonucleotides attached to magnetic beads. Unlike CROP-seq, direct-capture Perturb-seq constructs do not have a specific 5’ primer landing site and use template switch oligo (TSO) present on all amplified cDNA. Nextera reverse primer can also distinguish direct-capture Perturb-seq from CROP-seq constructs, which are amplified with Illumina reverse primer. Fig. 6B show's bioanalyzer traces of epigenetic factor library' in direct-capture Perturb-seq format amplified from TSO for full-length (FL) sequences or from primer specific to 3’ end barcode (BC). Percentage of cells identified as expressing none, single, or multiple pre-crRNA constructs. Signal from representative pre-crRNA obtained by full-length and barcode detection methods. Fig. 6C is the same as in Fig. 6B but for a set of pre-crRNAs in direct -capture Perturb-seq format targeting the cell-surface receptors. Fig. 6D depicts the construct scheme for direct-capture CROP-seq format with two vignettes differentiating between amplification of the same RNA detected with capture sequences from polyA:polyT interaction. Fig. 6E shows bioanalyzer traces, percentage of cells and signal from representative pre-crRNA obtained from direct-capture CROP-seq constructs amplified from capture-sequences as shown in the opposite scheme in Fig. 6D. Fig. 6F is the same as in FIG. 6E but for the amplification from polyA:polyT interaction. Fig. 6G shows the addition of specific forward amplification primer to the 5 ’ of pre-crRNA in direct-capture Perturb-seq format. Fig. 6H shows the bioanalyzer trace and signal from pre-crRNAs in the format outlined in Fig. 6G. Fig. 61 shows the bioanalyzer trace and signal from pre-crRNAs in CROP-seq format run alongside the pre-crRNAs shown in h. I, = ladder, * marks a band for dual-guide constructs, ** - quad-guide constructs.

[0038] Figs. 7A-7B show western blot of Casl2a levels upon dTagVl addition to induce its degradation. Fig. 7A shows H358 cells expressing enAsCas12a without FKBP12F36Vdomain show no degradation of Casl2a with IpM dTagVl treatment. Sample labeled ‘No’ was treated with DMSO. Fig. 7B show's H358 cells expressing enAsCas12a fused to FKBP12F36V(Casl2a-F36V) show progressive degradation of Casl2a with IpM dTagVl treatment. Casl2a disappears to levels below detection after 6 hours incubation with the chemical. Sample labeled ‘No’ was treated with DMSO.

[0039] Figs. 8A-8D show' full length pre-crRNA counts. Fig. 8A shows the percentage of cells identified as expressing none, single, or multiple pre-crRNA constructs from full-length detection method. Fig, 8B is a signal from representative pre-crRNA. Fig. 8C shows a boxplot of UMI signals for all pre-crRNA in the library'. Statistical comparison between samples was performed by a Mann-Whitney U test whereby the p-values were 0 (Casl2a), 1.57e’78(24 hours), and 0.9193 (48 hours), as compared to the first sample (no Casl2a), Fig.

[0040] 8D shows the percentage of cells with consistent pre-crRNA identify calls between full- length and barcode methods after 24 hours or 48 hours dTagVl treatment overlaid over previously shown data from cells with and without Casl2a. Degradation of Gas 12a restores consistency in cell identity calls to the levels of cells without Casl2a.

[0041] Fig 9 shows the pre-crRNA counts in K562 cells. Boxplot of UMI signals for pre-crRNAs obtained from CROP-seq vectors expressed in K562 cells. Similar to H358 cells, degradation of Cas 12a recovers pre-crRNA counts. Statistical comparison between samples was performed by a Mann-Whitney U test whereby the p-value was 1,2e"278.

[0042] Figs. 10A-10B show' epigenetic factor screen details. Fig. 10A is a heatmap of gene expression changes in response to genetic perturbations from Perturb-seq data. Values correspond to log fold changes in expression of profiled genes (columns, g=12,522) resulting from various genetic perturbations (rows). The dendrogram on the x-axis clusters perturbations with similar gene expression profdes, while the dendrogram at the top clusters genes with similar response patterns across perturbations. KDM1A and NATIO perturbations are highlighted in the main text with the additional examples following here. Fig. 10B shows the ribosomal proteins commonly downregulated among several perturbations as highlighted on the heatmap including cells with NATIO pre-crRNA featured in Figs. 2A-2I.

[0043] Figs. 11A-11F depict Casl2a CRISPRi details. Fig. 11A shows a scheme showing Casl2a CRISPRi constructs used; dead enAsCasl2a (denAs), multiAsCasl2a (multiAs), dead HyperLbCasl2a (HyperLb). Fig. I IB show's the experimental procedure. Fig. 11C depicts quantification of FACS data obtained from comparing Casl2a CRISPRi denAs, multiAs, and HyperLb in K562 and DLD Is where NTC = non-targeting control. Error bars represent SD Statistical comparison between guide formats was performed by an unpaired two-sample t-test with Welch’s correction whereby *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 as compared to the NTC. Fig. 1 ID depicts experimental procedure with dTagVl Figs. 1 IE-1 IF depict quantification of FACS data (Fig. 1 IE.) and qPCR data (Fig, 1 IF) obtained from TIyperLb-Casl2a CRISPRi in K562 and DLD Is at days 7 and 14 with the addition of dTagVl for 24, 48, and 72 hours. Error bars represent SD. Statistical comparison between guide formats was performed by an unpaired two-sample t-test with Welch’s correction whereby *p < 0.05, **p < 001, ***p < 0.001, ****p < 0.0001 as compared to the NTC.

[0044] DETAILED DESCRIPTION

[0045] Overview

[0046] The rapidly evolving CRISPR toolbox has transformed functional genomics by offering efficient, versatile, and complementary approaches for high-throughput genetic modification CRISPR-based functional genomics has enabled systematic, large-scale intenrigation of gene function in diverse biological systems. Toe associated technology platforms have been instrumental ftw identifying regulators of proliferation, drug resistance, and cell fate. Efforts have been made to broaden the application of functional genomic screens through targeting of multiple genes in each ceil and / or to simplify the experimental process by reducing the number of constructs used for a screening library CRISPR-associated protein 12a (Casl2a, previously Cpfl) has emerged as a useful nuclease for these purposes. As with Cas9 and other Class II CRISPR enzymes, Casl2a can be programmed with a short guide RNA (crRNA) to direct site-specific DNA breaks. However, unlike Cas9, it has a native ability to process an expressed guide RNA array (pre-crRNA). This minimizes the burden of cloning plasmid libraries intended to express several independent Cas12a crRNA sequences from each vector, which can then disrupt one or many genes at a time. Engineered variants of Casl 2a that boost specificity and activity have further enhanced its capabilities as a gene perturbation technology

[0047] lire power of functional genomic screens has dramatically increased through direct integration of CRISPR-mediated gene perturbation and single-cell (sc) assay technologies (e.g. scRNA-seq), an approach which has since been broadly termed Perturb-seq. While traditional pooled CRISPR. screens rely on simplified phenotypic outputs such as survival or reporter activity, Perturb-seq enables researchers to link multiple and specific gene modjficarions to transcnptotnic profiles and informa tion -ri ch phenotypes at cellular resolution. Upon delivery of guide RNAs into a cell population, expression changes for thousands of mRNAs in response to numerous individual gene-targeting events can be measured with single cell resolution. This enabled detection of rich and heterogeneous cellular phenotypes that went beyond individual pathway reporters or overt viability effects used for standard, pooled vector-based screens.

[0048] Despite ongoing advances in Perturb-seq methodologies, Perturb-seq has primarily relied on Cas9 for gene targeting, which imposes challenges for combinatorial gene editing and compact library' design. This is particularly problematic for Perturb-seq, where the number of cells that can be assayed per sequencing run limits the scale of screening experiments, making efficient use of each perturbation essential. Cas9-based systems typically require a separate Pol III promoter (e.g., U6) for each gene-specific guide RNA, rendering multi-guide constructs labor-intensive to assemble and expensive to produce at scale, hr contrast, Casl2a (Cpfl) processes a single transcript encoding multiple pre-crRNAs into individual guide RNAs, enabling expression of multi -target array s from a single promoter [ref]. The use of Casl 2a streamlines cloning, reduces synthesis costs, and permits delivery of multiple guides targeting the same gene, or distinct genes, from comparatively few constructs. The result is a more scalable and cost-effective framework for high- confidence individual or combinatorial perturbations using compact, information-rich libraries well suited to the resolution of single-cell functional genomics.

[0049] To date, Casl2a has yet to be applied for Perturb-seq, likely due to technical barriers related to linking (pre-)crRNAs to single-cell transcriptomes. In standard applications, Casl 2a and, in particular, its pre-crRNA array are delivered using lentiviral vectors that integrate into the genome of target cells. WTiile the guide RNAs are transcribed from a Pol III promoter to ensure functional activity, a separate Pol Il-derived mRNA is also transcribed from the lentiviral backbone. This transcript includes both the selectable marker and upstream elements, including the Pol III promoter and the pre-crRNA array. We hypothesize that Casl 2a cleaves the pre-crRNA sequence within this Pol II transcript as part of its normal guide processing activity, destabilizing the RNA and preventing its capture during single-cell RNA sequencing.

[0050] To harness the unique multi-guide expression capabilities of Casl 2a for single-cell applications, a comprehensive suite of pre-crRNA and “enhanced” Acidaminococcus sp. (enAs)Cas!2a expression vectors were developed, as discussed herein, along with refined methods for sequencing library preparation and analysis. Tire chemically-controlled “enhanced” Acidaminococcus Casl2a (enAsCas!2a) variant fused to an FKBP12F36V degron described herein allowed acute degradation of the nuclease prior to transcriptome profiling. This preserved the integrity of Pol Il-derived transcripts containing the guide array, enabling accurate perturbation assignment. Further described herein are optimized guide cassette designs and implementation of complementary recovery' strategies to improve guide detection efficiency. In a proof-of-concept Perturb-seq screen targeting chromatin regulators, an exemplary approach descnbed herein enabled efficient gene disruption and robust assignment of perturbation-specific transcriptional signatures across thousands of single cells. Tire effective targeting of at least four genes from a single vector was demonstrated and used to identify transcriptomic signatures that resulted from the disruption of select epigenetic factors using a custom, minimal pre-crRN A library'.

[0051] In parallel, additional opportunities for Casl2a-based loss-of-function screening was assessed through the development of improved transcriptional repressors. For this, a chemically -degradable “Hyper” Lachnospiraceae Casl2a (HyperLbCasl2a) fused to the ZIM3 / KRAB repressor domain was developed. The resulting variant, HyperLbCasl2a-ZIM3, achieved near-complete suppression of up to three targe t genes per cell, outperforming previously described Casl2a-based repressors across evaluated targets and cell types The silencing effect was rapidly reversed upon editor depletion. Nevertheless, the HyperLbCasl2a-ZIM3 system provided a potent, modular tool for multiplexed epigenetic repression, particularly for circumstances where transient, tunable silencing may be advantageous.

[0052] Together, this new next-generation Casl2a platform expanded the scalability, efficiency, and flexibility of Perturb-seq screening, thereby enabling increased complexity of future Perturb-seq screening campaigns, while opening new avenues for programmable gene silencing in complex cellular contexts.

[0053] Some aspects of the technology relate to methods for the assessment of perturbations with single cell molecular resolution. In some embodiments, methods are used for multiplexed assessment of perturbations with single cell molecular resolution. Multiplexed assessment can include evaluating multiple parameters and / or samples (e.g, cells) simultaneously within a single experiment or process, for example. One such example of a multiplexed assessment is Perturb-seq,

[0054] Perturbation Sequencing

[0055] Perturb-seq, or Perturbation Sequencing, is a technique that combines CRISPR-based gene perturbations with single-cell RNA sequencing (scRNA-seq) to, for example, study the effects of gene perturbations on transcriptional profiles at the single-cell level. Tire technique generally begins with the design and cloning of a library' of single-guide RNAs (sgRNAs) targeting genes of interest, along with appropriate control sgRNAs, The sgRNA library is typically introduced into cells using a delivery' vehicle, for example, a lenti viral or other retroviral delivery system, ensuring efficient and stable integration. Other delivery vehicles are described elsewhere herein. Cells are then transduced with the viral particles at a low multiplicity’ of infection (MOI) to ensure each cell receives, on average, one sgRNA. A selection marker, such as antibiotic resistance or fluorescent protein expression, for example, can be used to enrich for successfully transduced cells.

[0056] Following transduction, cells are cultured under conditions that allow the gene perturbations to take effect. After an appropriate incubation period, single-cell gene expression analysis, such as single-cell RNA sequencing, is performed. For example, single cells can be encapsulated into microfluidic droplets or captured using a microplate -based system, Within these compartments, cellular RNA can be reverse-transcribed into cDNA, and a cell-specific barcode is added to each transcript. The cDNA can then be amplified and subjected to high-throughput sequencing. Alongside the transcriptomic data, the sgRNA expressed in each cell can also be sequenced, enabling the identification of a specific perturbation associated with each cell.

[0057] The resulting dataset can be analyzed to determine how gene perturbations alter transcriptional programs. This can involve aligning sequencing reads to a reference genome, associating sgRNAs with their corresponding cells, and performing differential gene expression and pathway analyses. The perturbation-induced transcriptional changes can provide insights into gene function, gene regulatory' networks, and the mechanisms underlying specific biological processes or diseases, for example.

[0058] CROP-seq also combines CRISPR-based gene perturbation with single-cell RNA sequencing (scRNA-seq) to study the transcriptional consequences of genetic modifications at single-cell resolution. This technique generally begins with a pooled CR1SPR gRNA library in a modified (e.g., lentiviral) CROP-seq vector. The vector can include, for example, a U6 promoter for gRNA expression and, in some instances, an MS2 sequence for embedding the gRNA within the transcriptome for subsequent sequencing. After validating the library; lentivirus can be produced by transfecting the CROP-seq vector, along with packaging and envelope plasmids, into HEK293T cells or other cells, for example, those disclosed elsewhere herein. Hie viral particles can then be harvested and concentrated.

[0059] Next, the target cells (e.g., primary cells or cell lines) can be transduces with the lentivirus (e.g., at a low MOI) to ensure that most cells receive, on average, a single gRNA. The transduced cells can then be expanded to enable phenotypic stabilization following gene perturbation, for example.

[0060] Once cells are ready for analysis, they can be processed for single-cell RNA sequencing or other similar gene expression technique. For example, individual cells can be isolated using droplet-based microfluidics platforms (e.g., lOx Genomics Chromium) to capture and barcode individual cells along with their transcriptomes. During library¬ preparation, the embedded gRNA sequence can be transcribed as part of the polyadenylated transcriptome and can captured along with cellular RNA. Sequencing libraries can then be prepared and sequenced using a high-throughput next-generation sequencing platform, for example.

[0061] Computational tools can then be used to align scRNA-seq reads, identify gRNA sequences, and integrate gRNA assignments with transcriptome profiles. Downstream analysis involves, in some embodiments, identifying differential gene expression, clustering cells based on perturbations, and linking perturbations to phenotypic changes, for example. Regulatable Cas Enzymes with Intrinsic RNase Activity

[0062] Aspects of the technology include regulating pre-crRNA processing activity of a Cas enzyme with intrinsic RNase activity. Tire Cas enzymes used herein are modified, in some embodiments, such that they are regulatable, for example repressible or activatable. Thus, in some embodiments, methods herein include repressing pre-crRNA processing activity of a repressible Cas enzyme with intrinsic RNase activity (e.g,, repressible Casl2a enzyme). In addition to having target DNA cleavage activity, a Cas enzyme, such as Cas 12a, can process precursor CRISPR RNAs (pre-crRNAs) into mature crRNAs without the need for additional proteins. Contrast this to Cas9, which is not able to process its own crRNA and instead relies on trans-activating crRNA (tracrRNA) and RNase III. Cas 12a (formerly known as Cpfl) is a CRISPR-associated endonuclease with unique properties compared to Cas9, particularly its ability to process its own CRISPR RNA (crRN A). This RNase activity is facilitated by a conserved RuvC-like domain within the Casl2a protein. Other examples of Cas enzymes that have intrinsic RNase and crRNA processing activity include Casl2b, CasI2c, Casl2d, Casl2e, Casl2f, Casl2g, and variants thereof.

[0063] In some embodiments, a regulatable Cas enzyme with intrinsic RNase activity is a Casl 2a enzyme. Non-limiting examples of Casl2a enzymes include AsCasl2a (Acidaminococcus sp.) enzymes, LbCasl2a (Lachnospiraceae bacterium) enzymes, FnCasl2a (Francisella novicida) enzymes, Mb2Casl2a (Moraxella bovoculi) enzymes, EnCasl2a (Eubacterium eligens) enzymes, BbCasl2a (Bacillus hisashii) enzymes, and variants thereof. In some embodiments, a Cas 12a enzyme is an AsCasl2a enzyme. In some embodiments, a Cas 12a enzyme is an enhanced AsCas 12a enzyme, for example, comprising the ammo acid sequence of SEQ ID NO: 2.

[0064] In some embodiments, a regulatable Cas enzyme with intrinsic RNase activity comprises an amino acid sequence having at least 85 % (e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to a wild-type Cas enzyme with intrinsic RNase activity (e.g., any of the Cas enzymes having intrinsic RNase activity described herein). In some embodiments, a regulatableCas enzyme with intrinsic RNase activity comprises an amino acid sequence having at least 85% (e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to a wild-type Cas 12a enzyme (e.g,, any of the CasI2 enzymes described herein). In some embodiments, a Cas 12a enzyme comprises an amino acid sequence having at least 85% (e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the amino acid sequence of SEQ ID NO: 2. The Cas enzymes used herein, in some embodiments, are “regulatable'’ Cas enzymes having intrinsic RNase activity’ (e.g., repressible Casl2a). A regulatable enzyme includes enzymes, the expression or activity of which can be activated (induced) or repressed (partially or completely inhibited) in response to a specific regulatory signal (an inducer or inhibitor). In some embodiments, a regulatable Cas enzyme with intrinsic RNase activity (or a nucleic acid encoding such an enzyme) is linked to a regulatory’ domain, for example, a DNA-binding domain, a ligand-binding domain, an effector-binding domain, an allosteric regulatory’ domain, a signal transduction domain, a transcriptional activation domain, a post- translational modification (ptm) domain, or an autoinhibitory domain.

[0065] Tire Cas enzymes used herein, in some embodiments, are “repressible” Cas enzymes having intrinsic RNase activity (e.g., repressible Casl2a enzymes). A repressible enzyme includes an enzyme the expression or activity’ of which can be decreased or completely inhibited in response to a specific regulatory signal. In some embodiments, a repressible Cas enzyme with intrinsic RNase activity (or a nucleic acid encoding such an enzyme) is linked to a repressible domain. Non-limiting examples of repressible domains include degron domains, N-degrons (destabilizing residues, such as Arg, Lys, Pho), ubiquitination-related domains (e.g,, PEST sequences, D-box, KEN Box, SOCS Box), phosphor-degrons (p-catenin phospho-degron), lysosomal targeting signals (e.g., KFERQ motif, LAMP-2A binding motifs, ubiquitin-like tags), protease recognition sites (e.g., caspase cleavage sites, matrix metalloprotease (MMP) recognition sequences), SUMOylation sites (e.g., SIM (SUMO Interacting Motif)), steric hindrance or autoinhibitory’ domains (e.g., PELP1 degron-like motif, IDRs (Intrinsically Disordered Regions), and other regulatory domains, such as F-box Domains, HIM (u biquitin-interacting motif), and zinc-fmger or leucine-rich motifs. In some embodiments, a repressible Cas enzyme (e.g, a repressible Cas12a enzyme) comprises a degron domain. In some embodiments, the degron domain is a C-terminal FKBP K336' fusion domain. Thus, in some embodiments, a method herein comprises culturing a population of cells in the presence of a dTag small molecule dTag VI, following a culturing period sufficient to effect perturbation by the enzyme.

[0066] Activity of Cas enzymes with intrinsic RNase activity (e.g., Cas 12 enzymes) can be regulated using other means. For example, ProteoTuner technology makes use of a 12-kDa ligand-dependent destabilization domain (DD), derived from an unstable FKBP12 mutant, that is fused to a protein of interest (e.g., at the N- or C -terminus) by targeting it for proteasomal degradation. Addition of a membrane-permeant small molecule (750 Da) ligand, Shieidl, reversibly stabilizes the fusion protein by protecting it from being degraded, allowing the fusion protein to accumulate in the cell. Thus, the stability of a Cas 12 enzyme can be controlled by fusing it to a DD tag and adding or removing Shieldl to modulate stability of the Casl2 fusion.

[0067] In some embodiments, the RNA processing activity of a repressible Cas enzyme with intrinsic RNase activity (e.g., a repressible Casl2a enzyme) is decreased or completely inhibited in response to a specific regulatory signal. Non-limiting examples of regulatory signals include hormones, growth factors, small molecules, transcription factors, and environmental stimuli, or a combination thereof. Selection of the regulatory signal depends, at least in part, on how the repressible target has been modified to become repressible.

[0068] Modification to induce repression can include, for example, linking a repressible pre-crRNA processing Cas enzyme to a repressible domain, such as a degradation tag. Non-limiting examples of degradation tags include degradation tag (dTAG), auxin -inducible degron (AID), HaloTag, and SMASh. In some embodiments, the specific regulatory signal includes a small molecule. A small molecule may be selected from a group including dKl, auxin, asunaprevir, or combinations thereof. Upon addition of the small molecule, the activity of a repressible Cas enzyme with intrinsic RNase activity is decreased or completely inhibited. In some embodiments, this includes reducing or completely inhibiting the RNase activity of a repressible Cas enzyme with intrinsic RNase activity (e.g., Casl2a, Casl2b, Casl2c, Casl2d, Cas 12e, Casl2f, Casl2g, and variants thereof) allowing for detection of pre-crRNAs.

[0069] Deactivated (Dead) Cas Enzymes

[0070] In some embodiments, a regulatable Cas enzyme with intrinsic RNase activity is a deactivated Cas enzyme (dCas enzyme). Deactivated Cas enzymes include variants of Cas proteins that have been engineered to lose their DNA-cleaving activity. This is achieved by introducing mutations in the catalytic domains responsible for cutting DNA. For example, in Cas9, mutations in the RuvC and HNH nuclease domains render the protein catalytically inactive. As a result, dCas enzymes retain their ability to bind to specific DNA sequences, guided by a complementary guide RNA, but cannot introduce DNA strand breaks.

[0071] Deactivated Cas enzymes can be used, in some instances, for CRISPR interference (CRISPRi) techniques or CRISPR activation (CRISPRa) techniques, in accordance with the present disclosure.

[0072] In some embodiments, a regulatable (e.g., repressible) dCas enzyme with intrinsic RNase activity can be fused to a transcriptional repressor to block transcription by sterically hindering RNA polymerase or altering chromatin structure, for example This allows precise and reversible gene knockdown without permanently modifying the DNA sequence, for example. In some embodiments, a repressible dCas enzyme (e.g., repressible dCasl2a enzyme) is linked to a transcription repressor domain, for example, selected from KRAB (Kriippel-associated box), SID (Sin3 Interaction Domain), MeCP2 (Methyl-CpG-Binding Protein 2), ESDI (Lysine-Specific Demethylase 1), dCas9-Mxil, CoREST, Ezh2 (Enhancer of Zeste Homolog 2), HP1 (Heterochromatin Protein I), REST (Repressor Element-1 Silencing Transcription Factor), Polycomb Repressor Complex (PRC2), ZIM3 (Zinc Finger Imprinted 3), DNMT3A (DNA methyltransferase 3 alpha), and DNMT3L (DNA methyltransferase 3 like).

[0073] In some embodiments, a regulatable (e g., repressible) dCas enzyme with intrinsic RNase activity can be fused to a transcriptional activator to enhance the expression of target genes. In some embodiments, a repressible dCas enzyme (e.g., repressible dCas!2a enzyme) is linked to a transcription activation domain, optionally selected from VP64, p65, Rta (Replication and Transcription Activator), VPR (VP64-p65-Rta), SunTag, HSF 1 (Heat Shock Factor 1), P300 Core, SAM (Synergistic Activation Mediator), and ET1 (Epstein-Barr Virus EBNA-LP Transactivation Domain).

[0074] Pre-crRNA Arrays

[0075] Pre-crRNA (precursor CRISPR RNA) includes the primary, unprocessed RNA transcript Pre-crRNA serves as the initial RNA product that undergoes processing to produce mature crRNAs, which guide Cas proteins to target specific DNA or RNA sequences for cleavage. In the context of CRISPR-Casl2a gene editing, for example, pre-crRNA (precursor CRISPR RNA) is a transcribed RNA molecule that serves as the initial guide RNA before being processed into its fimctional form, Cas 12a, among other Cas enzymes with intrinsic RNase activity, has the ability to process its own pre-crRNA into mature crRNA without requiring additional enzymes. This self-processing capability simplifies the design of multiplexed gene editing systems, where multiple target sites can be addressed using a single pre-crRNA transcript.

[0076] In some embodiments, a pre-crRNA includes a series of direct repeats interspersed with spacers. Repeats (also referred to as direct repeat domains) include short, conserved sequences recognized by the Cas enzyme for processing, while spacers include unique sequences complementary to a target DNA (e.g., target gene), determining the specificity of DNA binding and cleavage. A pre-crRNA is generally a single RNA molecule transcribed as a long array, typically including multiple repeat-spacer units when multiplexing, for example. Ihe Cas enzyme recognizes the repeat sequences within the pre-crRNA and cleaves them to release individual mature crRNAs, each containing a single spacer flanked by partial repeat sequences.

[0077] In some embodiments, a crRNA of a pre-crRNA array comprises, in the following order, a direct repeat (DR) domain, a Spacer domain, and a 3' end. In some embodiments, a crRNA of the pre-crRNA array comprises a capture sequence, for example, at the 3' end of each pre-crRNA.

[0078] In some embodiments, a method comprises delivering a pooled library of vectors, each encoding a pre-crRNA array comprising multiple crRNAs to the initial population of cells. A pooled pre-crRNA library includes a collection of pre-designed, pre-crRNAs encoding multiple guide RNAs (crRNAs).

[0079] In some embodiments, multiple crRNAs target a single genomic locus of interest. In some embodiments, multiple crRN As target multiple genomic loci of interest. Non-limiting examples of genomic loci that can be targeted by one or more crRNAs include introns, exons, promoters, enhancers (e.g, long-distance enhancers), non-coding RNAs, and topologically associating domains (TADs). In some embodiments, multiple crRNAs target a single gene of interest. In other embodiments, multiple crRNAs target at least two different regions of the single gene of interest. In yet other embodiments, multiple crRN As target two or more genes of interest.

[0080] Cell Transfection / Transduction

[0081] Methods of the disclosure, in some aspects, initially include delivering a vector encoding a pre-crRNA array comprising a barcode and multiple crRNAs to an initial population of cells, wherein cells of the initial population comprise a regulatable Cas enzyme with intrinsic RNase activity (e.g., a repressible Cas 12a enzyme). In some embodiments, delivering of the vector comprises transducing cells of the population with the vector such that single cells of tire initial population receive a single pre-crRNA array. In some embodiments, individual cells of the initial population of cells receive at least one perturbation, and the barcode of the pre-crRN A array comprises a sequence identifying the perturbation.

[0082] In some embodiments, an initial population of cells is cultured to effect Cas enzyme (e.g., Cas 12a enzyme) -mediated perturbation, thereby producing a population of perturbed cells. Cell culture, in some embodiments, involves various techniques in which cells are grown in an artificial, controlled environment. The artificial, controlled environment generally involves exposing the cells to growth medium and, optionally, various forms of growth factors, antibiotics, or antimycotics, the combination of which is changed periodically by manual or automatic manipulation. Culturing cells in this way allows for normal biological activity to be maintained ex-vivo, thereby mimicking how cells may behave within a living organism. Normal biological activity can include, for example, maintenance of cell proliferation, cell differentiation, signal transduction, and enzymatic activity. In some embodiments this includes degradation of pre-crRN As within each cell of the initial population by the Cas9 enzyme with intrinsic RNase activity (e.g., Casl 2a, Casl2b, Casl2c, Casl 2d, Casl 2e, Casl2f, Casl 2g, and variants thereof). This can result in a population of perturbed cells with substantially fewer pre-crRNAs. The methods described herein can be used to repress the Cas enzyme with intrinsic RNase activity following genetic or epigenetic perturbation, thus increasing the barcoded crRNAs available for analysis.

[0083] Delivery Vehicles

[0084] Vectors include delivery vehicles used to introduce an agent (e.g., genetic material) into target cells, for example through transfection or transduction, In some embodiments, a vector is a viral vector (e.g., engineered virus or viral genome). Non-limiting examples of viral vectors include lentiviral vectors, adenoviral vectors, adeno -associated viral (AAV) vectors, retroviral vectors, herpes simplex virus (HSV) vectors, sendai virus (SeV) vectors, and baculovirus vectors.

[0085] A vector, in some aspects, encodes a pre-crRNA array comprising multiple crRNAs. It should be understood that the phrase “vector encoding" includes a “vector comprising a nucleic acid encoding."" Pre-crRNA (pre-CRISPR RNA) includes a precursor RNA molecule transcribed as a single long RNA strand from a CRISPR array, which can be composed of alternating repeat and spacer sequences. It can serve as the precursor to mature crRNA (CRISPR RNA), which guides the CRISPR-Cas complex to recognize and cleave complementary sequences in foreign DNA or RNA. Pre-crRNA undergoes processing by Cas enzymes having pre-crRNA processing activity also known as intrinsic RNase activity, such as Casl2a enzyme. This cleavage produces individual crRNA units, each containing a spacer-derived sequence that determines target specificity and a portion of the repeat sequence, which may form secondary' structures required for binding to Cas proteins.

[0086] In some embodiments, a vector is lentiviral vector. The core components of a lentiviral vector system typically include a transfer vector, packaging plasmid, and envelope plasmid. A transfer vector, referred to herein as a “lentiviral vector,” includes a nucleic acid encoding a pre-crRNA or a Casl2a enzyme, which is driven by a promoter, such as U6 or EFla, for example. A lentiviral vector can also contain long terminal repeats (LTRs) at the 5’ and 3’ ends, which are for viral replication and / or integration. Other elements within the lentiviral vector can include a psi ( ) packaging signal, which enables RNA genome recognition and incorporation into viral particles, and regulatory' elements such as the woodchuck hepatitis virus post-transcriptional regulatory / element (WPRE) to enhance transgene stability and expression, along with a polyadenylation (poly A) signal for efficient transcription termination. A packaging plasmid encodes the structural and enzymatic components for viral particle assembly and function. This includes, for example, the gag gene, which provides structural proteins such as capsid and matrix proteins; the pol gene, which encodes enzymes such as reverse transcriptase and integrase for genome replication and integration; and, in some embodiments, the rev gene, which facilitates the export of unspliced viral RNA from the nucleus to the cytoplasm. An envelope plasmid provides the viral envelope protein, which determines the vector’s tropism, or cell type specificity. A commonly used envelope protein is vesicular stomatitis virus G (VSV-G), for example. Alternative envelope proteins can also be used. In some embodiments, lentiviral vectors are produced by co-transfecting HEK293 cells (e.g., HEK293T or HEK293F cells), Vero cells, HT1080 cells, or other cells with the plasmid components. The cells package the lentiviral vector RNA into viral particles, which can then be collected for use as described herein.

[0087] In other embodiments, a vector is a non-viral vector. Non-limiting examples of non-viral vectors include plasmid DNA, minicircle DNA, transposons, and artificial chromosomes.

[0088] Incubation and Perturbation

[0089] Methods of the disclosure, in some aspects, include culturing an initial population of cells to effect Casl2a enzyme-mediated perturbation, thereby producing a population of perturbed cells. In some embodiments, methods herein comprise delivering a vector encoding a pre-crRNA array comprising a barcode and multiple crRNAs to an initial population of cells. An initial population of cells is simply a population of (e.g., two to more) cells (e.g., primary or cell line) into which a vector encoding a pre-crRNA is delivered (e.g., transfected or transduced). An initial population of cells should not be limited by the number of cells in the population. An initial population of cells (or any population of cells herein) can comprise, tens, hundreds, thousands, or millions of cells, or more. For example, a population of cells can comprise 10 to 10,000,000 cells. In some embodiments, a population of cells comprises 10 to 1,000,000; 10 to 100,000; 10 to 10,000; 10 to 1000; 100 to 10,000,000; 100 to 1,000,000; 100 to 100,000; 100 to 10,000; 100 to 1000; 1000 to 10,000,000; 1000 to 1,000,000; 1000 to 100,000; 1000 to 10,000; 10,000 to 10,000,000; 10,000 to 1,000,000; or 10,000 to 100,000 cells. In some embodiments, a population of cells (e.g., an initial population of cells) comprises at least 100, at least 1000, at least 10,000, at least 100,000, at least 1,000,000, or at least 10,000,000 cells.

[0090] In some embodiments, a population of cells is cultured for about 3 days to about 7 days to allow sufficient time for gene perturbation to occur. In some embodiments, a population of cells is cultured for about 3 days to about 5 days. In some embodiments, a population of cells is cultured for at least 3 days.

[0091] In the context of the present disclosure, perturbations include modifications introduced into a cell, for example, into one or more loci in a genome (e.g, within a gene or other genetic element modulating gene expression) of a cell (a genetic perturbation) or into the epigenetic landscape of cell (an epigenetic perturbation). For example, genetic perturbations can be used to assess gene function, while epigenetic changes can be used to assess gene regulation without altering the underlying DNA sequence Thus, in some embodiments, methods herein are for multiplexed assessment of genetic perturbations, while in other embodiments, methods herein are for multiplexed assessment of epigenetic perturbations. In yet other embodiments, methods herein are for multiplexed assessment of genetic perturbations and epigenetic perturbations.

[0092] In some embodiments, the genetic perturbations are selected from deletions, insertions, and substitutions. A deletion includes the removal or loss of a segment of DNA containing a specific gene from the genome. Gene deletion typically results in the absence of the gene product (e.g., a protein or RNA). An insertion includes the introduction of a specific DNA sequence, such as a gene or genetic element, into a gene (e.g., within a genome) at a particular location. A substitution includes the replacement of a specific DNA sequence with another DNA sequence.

[0093] In some embodiments, the epigenetic perturbations are selected from methylation (addition of a methyl group to a molecule), demethylation (removal of a methyl group to a molecule), acetylation (addition of an acetyl group to a molecule), deacetylation (removal of an acetyl group to a molecule), phosphorylation (addition of a phosphate group to a molecule), and dephosphorylation (removal of a phosphate group to a molecule) Following the cell culture period during which gene perturbations take effect, tire regulatable Cas enzyme with intrinsic RNase activity is repressed such that it cannot degrade crRNA needed to analyze the gene perturbation.

[0094] Single-Cell RNA Sequencing (scRNA-seq) and Data Analysis

[0095] Methods of the disclosure, in some aspects, include analyzing RNA sequences of individual perturbed cells (e.g., genetic and / or epigenetic perturbations) of the population of perturbed cells at single cell molecular resolution using a single-cell gene expression technique. Single cell molecular resolution includes an analysis at the level of individual cells --for example, the ability to analyze and quantify molecular characteristics (e.g., DNA, RNA, proteins, or metabolites) at the level of individual cells.

[0096] RNA-seq (RNA sequencing), in particular, is a high-throughput sequencing technique used to analyze the transcriptome, the complete set of RNA molecules (including mRNA, noncoding RNA, and other RNA species) present m a cell at a given time, it provides a detailed and quantitative view of gene expression, alternative splicing, RNA editing, and other tran scriptom ic features.

[0097] In some embodiments, RNA-seq begins with the extraction of RNA from the cells or tissues of interest. Once RNA is prepared, it is converted into complementary DNA (cDNA) through reverse transcription using a reverse transcriptase. This cDNA acts as a stable, amplifiable template for downstream processes. Next, a library' is prepared, whereby adapter sequences are added to the ends of the cDNA fragments to make them compatible with high-throughput sequencing platforms. The library is then amplified, for example, using polymerase chain reaction (PCR) to ensure there is sufficient material for sequencing. The resulting cDNA librai ' represents the transcriptome of tire original RNA sample. The prepared cDNA librai ' can then be subjected to sequencing using next-generation sequencing (NGS) platforms such as Illumina, PacBio, or Oxford Nanopore, for example. Tire sequencing reads are then processed and aligned to a reference genome or transcriptome to determine the origin of each read. Quantification algorithms can be used to, in some instances, measure the abundance of transcripts, providing a comprehensive view of gene expression levels.

[0098] Confirming perturbation and perturbation efficiency can be achieved in a number of ways including, for example, RNA-seq or qPCR (for transcriptional changes), Western blot or flow cytometry' (for protein depletion / activation), and / or functional assays. In some aspects, methods herein further comprise determining measured differences in the RNA sequences that are correlated to perturbations detected for individual perturbed cells.

[0099] Compositions

[0100] Also provided herein are compositions compnsing a delivery vehicle comprising a nucleic acid encoding a regulatable Cas enzyme having intrinsic RNase activity and a delivery vehicle comprising a nucleic acid encoding a pre-crRNA array. In some embodiments, a composition comprises a vector (e.g., a lentiviral vector) encoding a repressible Cas 12a enzyme and a vector encoding a pre-crRNA array. In some embodiments, the composition comprises multiple vectors (e.g., hundreds or thousands of vectors), each encoding a pre-crRNA array.

[0101] Further provided herein are cells comprising a vector encoding a pre-crRNA array, wherein the cell expresses a regulatable Cas enzyme with intrinsic RNase activity. In some embodiments, the regulatable Cas enzyme is a repressible Cas 12a enzyme.

[0102] Additional Embodiments

[0103] Additional embodiments of the technology are described in the following numbered paragraphs:

[0104] 1. A method for multiplexed assessment of perturbations with single cell molecular resolution, the method comprising:

[0105] (a) delivering a vector encoding a pre-crRN A array comprising a barcode and multiple crRNAs to an initial population of cells, wherein cells of the initial population comprise a regulatable Cas enzyme with intrinsic RNase activity;

[0106] (b) culturing the initial population of cells to effect Cas enzyme-mediated perturbation, thereby producing a population of perturbed cells;

[0107] (c) repressing pre-crRNA processing activity of the regulatable Cas enzyme; and (d) analyzing RNA sequences of individual perturbed cells of the population of perturbed cells using a single-cell gene expression technique. 2. The method of paragraph 1, wherein individual cells of the initial population of cells receive at least one perturbation, and the barcode of the pre-crRNA array comprises a sequence identifying the perturbation.

[0108] 3. lire method of paragraph 1 or 2, wherein the perturbations are selected from genetic perturbations and epigenetic perturbations,

[0109] 4. The method of paragraph 1, wherein the genetic perturbations and epigenetic perturbations are selected from deletions, insertions, and substitutions,

[0110] 5. The method of any preceding paragraph, wherein the multiple crRNAs target a single genomic locus of interest, optionally a gene of interest.

[0111] 6. The method of any preceding paragraph, wherein the multiple crRNAs target at least two different genomic loci of interest, optionally two or more genes of interest.

[0112] 7. Tire method of any of paragraphs 1 -4, wherein the multiple crRNAs target two or more loci within the same genes of interest.

[0113] 8. Th e method of any preceding paragraph, wherein each crRNA of the pre-crRNA array comprises, in the following order, a direct repeat (DR) domain, a Spacer domain, and a 3' end.

[0114] 9. Idle method of paragraph 7, wherein each crRNA of the pre-crRNA array further comprises a capture sequence.

[0115] 10. Idle method of paragraph 8, wherein the capture sequence is at the 3' end of each crRNA.

[0116] 11. The method of any preceding paragraph, wherein (a) comprises delivering a pooled library' of vectors, each encoding a pre-crRNA array comprising multiple crRNAs to the initial population of cells.

[0117] 12. The method of paragraph 11, wherein the regulatable Cas enzyme comprises a degron domain, optionally, wherein the degron domain is a C -terminal FKBP12E’bVfusion domain, and (c) comprises culturing the initial population of cells in the presence of a dTag small molecule dTagV 1.

[0118] 13. The method of any preceding paragraph, wherein the regulatable Cas enzyme is a repressible enhanced Acidaminococcus sp. Cas 12a (enAs)Cas 12a enzyme, optionally wherein the repressible (enAs)Casl2a enzyme comprises the amino acid sequence of SEQ ID NO: 1. 14. The method of any preceding paragraph, wherein the regulatable Cas enzyme is a regulatable deactivated Cas (dCas) enzyme,

[0119] 15. Hie method of paragraph 14, wherein the regulatable dCas enzyme is linked to a transcription repressor domain, optionally selected from KRAB (Kriippel-associated box), SID (Sin3 Interaction Domain), MeCP2 (Methyl-CpG-Binding Protein 2), LSD1 (Lysine-Specific Demethylase 1), dCas-Mxil, CoREST, Ezh2 (Enhancer of Zeste Homolog 2), HP1 (Heterochromatin Protein 1), REST (Repressor Element-1 Silencing Transcription Factor), Polycomb Repressor Complex (PRC2), ZTM3 (Zinc Finger Imprinted 3), DNMT3A (DNA methyl transferase 3 alpha), and DNMT3L (DNA methyl transferase 3 like).

[0120] 16. The method of paragraph 14, wherein the regulatable dCas enzyme is linked to a transcription activation domain, optionally selected from VP64, p65, Rta (Replication and Transcription Activator), VPR (VP64-p65-Rta), Sun Tag, HSF1 (Heat Shock Factor 1), P300 Core, SAM (Synergistic Activation Mediator), and ET1 (Epstein-Barr Virus EBNA-LP Transactivation Domain).

[0121] 17. Tlie method of any preceding claim, wherein the vector is a non-viral vector.

[0122] 18. The method of claim 17, wherein the non-viral vector is selected from plasmid DNA, minicircle DNA, transposons, and artificial chromosomes.

[0123] 19. The method of any of claims 1-16, wherein the vector is a viral vector.

[0124] 20. Tire method of claim 10, wherein the viral vector is selected from lentiviral vectors, adenoviral vectors, adeno-associated viral (AAV) vectors, retroviral vectors, herpes simplex virus (HSV) vectors, sendai virus (SeV) vectors, and baculovirus vectors

[0125] 21. The method of any one of paragraphs 17-20, wherein delivering of the vector comprises transducing cells of the population w ith the vector such that single cells of the initial population receive a single pre-crRNA array,

[0126] 22. The method of any preceding paragraph, wherein the single-cell gene expression technique comprises single-cell RNA sequencing,

[0127] 23. The method of any preceding paragraph further comprising determining measured differences in the RNA sequences that are correlated to perturbations detected for individual perturbed cells.

[0128] 24. Tlie method of any preceding paragraph, wherein the population of cells comprises primary cells and / or immortalized cells. 25. A method for multiplexed assessment of perturbations with single cell molecular resolution, the method comprising:

[0129] (a) culturing an initial population of cells comprising (i) a pre-crRNA array comprising a barcode and multiple crRNAs and (ii) a regulatable Cas enzyme with intrinsic RNase activity to effect Cas enzyme-mediated perturbation, thereby producing a population of perturbed cells; and

[0130] (b) repressing pre-crRNA processing activity of the regulatable Cas enzyme, 26. The method of paragraph 25 further comprising analyzing RNA sequences of individual perturbed cells of the population of perturbed cells using a single-cell gene expression technique.

[0131] SEQUENCES

[0132] enAsCas 12a_dTag MAKRTADGSEFESPKKKRKVGSGTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPI IDRI YKTYADQCLQLVQLDWENLSAAIDS RKEKTEETRNALIEEQAT PNAIHD FIGRTDNLTDAINKRZHAEI YKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYRNRKNVFSAEDISTAIPHRIVQDNFPKFKE NCHIFTRLITAVPSLREHFEN'’KIGAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLN EVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALF NELNSIDLTHIFISI-IKKLETISSA. LCDI-IWDTLRNALYERRISELTGKITKSJiKEKVQRSLKI-IEE'INLQEIISAAG KELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYZULLDWFAVDESNEVDPEFSARLTGI KLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLARGV7DVNREKNNGAILFVKNGLYYLGIMPKQKGRYKAL SFEPTEKTSEGFDKMYYDYFPEAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPK KFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAE KEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWYGLFSPENLAKYSIKLNGQA. ELFYRPKSPI'IKRMANRLG EKMLNKKLKDQKTPI PDTLYQELYDYVNHRLSHDLSDEARALLPNVI TKEVSHE I TKDRRFTSEKFFFHVP I TLN YQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVA ARQAWSWGTIKDLKQGYLSQVIHEIVDLMIHYQAVWLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVL KDYPAE GGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFWKTIKNHESRKrIFLEGFDF LHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENI-IRFTGRYRDLYP A ELIALLEEKGIVFRDGSNTLPKLLENEDSHAIDT ALIRSVLQMRNSNAATGEEYINSPVRDLNGVCFDSRF QNPEWPMEADA GAYHIALKGQLLLNHLKESKDLKLQNGI SNQDWLAYIQELRNAPKKKRKVGSAGSAA. GSGPKK KRKV DPKKKRKVGRAGGSG VQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEV IRGWEEGVAOMSVGQRAKLTISPDYAYGATGHPGIIPPI-IATLVFDVELLKLE* (SEQ ID NO:

[0133] 1) (Cas12a: FKBP12F36V: NLS)

[0134] Casl2a TQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLBWENL SAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTT TEHENALLRSFDKFTTYFSGFYRNRK TSAEDIST7IIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVK KAIGI FVSTS lEEVFSFPFYMQLLTQTQIDLYNQLLGGI SREAGTEKIKGLNEVLMLAIQKNDETAHI IASLPHR FIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNEWLETAEALFNELNSIDLTHIFISHKKLETISS ALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALD QPLPTTLKKQEEKEILKSQLDSLLGLYI-ILLDWFAVE’ESNEVDPEFSARLTGIKLEMEPSLSFYNIGiiRNYATKKPY SVEKFKLNFQMPTLARGWDVNREKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDA AKMI PKCSTQLKAVTAHFQTHTTP I LLSNNFI EPLE ITKE I YDLNNPEKEPKKFOTAYAKKTGDQKGYREALC IDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMEAVETGKLYLFQIYNKDFA KGHHGKPNLHTLYWTGLFSPENIAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQEL YDYVNHRLSHDLSDEARALLPWITKEVSHEI IKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPE TPI IGIERGER LIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVA ROATAISVVGTIKDLKQGYLSQVI HEIVDLMIHYQAVWLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTS FAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQ RGLPGFMP WDIVFEKNETQFD KGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILP KLLENDDSHAIDTWALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADAJIGAYHIALKGQL LLNHLKESKDLKLQNGISNQDWLAYIQELRN (SEQ ID NO: 2 )

[0135] Appendix A includes additional sequences and is herein incorporated by reference in its entirety.

[0136] EXAMPLES

[0137] Example 1: Optimization of pre-crRNA expression and capture for Perturb-seq To assess the potential for Casl2a in single-cell applications like Perturb-seq, a population of NCI-H358 (bronchioalveolar carcinoma) cells was generated to stably express a 4XNLS-enAsCas 12a transgene. Adaptations of the direct-capture and CROP-seq strategies for lenti viral delivery and subsequent single cell detection of pre-crRNAs were then explored (Fig, 3 A), For Cas9-based direct-capture, one of two different capture sequences (CS 1 or 2) was incorporated within a stem loop of the single-guide (sg)RNA scaffold or is appended to the sgRNA 3’ terminus. Due to differences between the Cas9-associated sgRNAs and the relatively shorter, reversed orientation of Casl2a-associated crR As, three different pre-crRNA configurations were tested with varying CS positions: at the 3 ’ end of the spacer (position 1), between the direct repeat (DR) and the spacer (position 2), and in the hairpin of the DR (position 3) (Fig. 3B). The effectiveness of each approach was tested through expression of a representative pre-crRNA array designed to simultaneously disrupt either the CD63 and CD47 or CD81 and CD9 genes (one crRNA per target, two targets per vector), respectively, followed by assessment of cell surface levels for each gene product (Figs. 3C-3D). The pre-crRNA engineered with a CS in position 1 showed comparable activity to an unmodified pre-crRNA, while CSs in the two other positions interfered with Cas12a targeting (Fig. 3E). Modest, context-dependent differences on targeting activity were observed with constructs incorporating CS1 vs CS2, suggesting capture sequence or experiment-specific effects could impact function.

[0138] Having established the optimal CS position, the efficacy of pre-crRNAs expressed from the direct-capture format was compared to otherwise unmodified pre-crRNAs within a CROP-seq vector, in the CROP-seq context, the pre-crRNA is adjacent to the lenti viral 3" long terminal repeat (LTR) and an RNA Pol-II termination sequence (Fig. 4A). This enabled the guides to be functionally expressed from an RNA Pol-III (U6) promoter formed from a duplication event during lentiviral integration. In addition, the pre-cRNA was incorporated into an RNA Pol-II EFl A, or EEFlAl)- smQ mRNA necessary for antibiotic (puromycin) selection marker expression. This mRNA is later amplified and the pre-crRNAs identified following oligo(d)T priming and 3’ scRNA-seq. The pre-crRNA is located too far away from die 3' LTR in the direct capture context for the guides to be identified using conventional 3' or 5' (i.e. SMART) scRNA-seq protocols. For relative assessment, pre-crRNAs were designed to be expressed from either the direct-capture Perturb-seq or CROP-seq formats for simultaneous, multiplexed disruption of either two (CD47 / CD63 or CD81 / CD9) or four genes (CD47 / CD63 / CD81 / CD9) (Fig. 4B).

[0139] Comparable perturbation of multiple targets resulted from each of the pre-crRNA vector contexts (Fig 4C). There was no overt difference in activity levels when individual crRNAs were reordered within the array, aldiough efficacy was impacted by the spacer sequence for a given target gene (Figs. 4C-4D).

[0140] There were no observable differences in targeting efficacy when comparing the CROP-seq and direct capture Perturb-seq guide RNA vectors expressing either two or four gene-specific crRNAs per vector, a substantial or consistent was not observed(Fig. 4B). However, it is possible that the dynamic range of those assays was limited due to the high efficiency of the selected guides. Also, while others have suggested there are minimal position effects for pre-crRNA arrays with <4 co-expressed guides, possibility with the single cell-formatted vectors through the use of these particular spacers could not be excluded.

[0141] To further assess differences in efficiency between vector formats and position within a pre-crRNA array, a set of pre-crRNAs w ns designed with the last position occupied by a new CD81 spacerthat induced relatively-low target depletion (Fig. 4C). A spacer with -50% efficiency would allow better distinction between crRNA expression contexts. Capture sequences were also added to the 3’ end of pre-crRNA in the CROP-seq vector, which created the direct-capture CROP-seq format.

[0142] The CD81 knock-out efficiency was measured with constructs expressing the less efficient spacer and again detected similar targeting efficiency betw een dual and quad pre-crRNA variants in both the CROP-seq and Perturb-seq formats (Fig. 4D).

[0143] Together, these data show that CROP-seq and direct-capture Perturb-seq formats exhibit comparable Casl2a-based target disruption with no significant differences between dual and quad pre-crRNA formats. In the direct-capture Perturb-seq format, it was determined that capture sequences (CS) at the 3 ’ end of the spacer performed best while other positions interfered with targeting. Additionally, knock-out efficiency was more influenced by the spacer sequences than by the pre-crRNA formats. Example 2: Casl2a impairs guide detection in pooled scRNA screens

[0144] Epigenetic modifiers have recognized roles in controlling gene expression and influencing cell fate changes, which can be monitored by scRNA -seq. Accordingly, a subset of these factors was selected for assessment with pooled enAsCasl2a CROP-seq or direct- capture libraries (Fig. 1A). For both formats, the vector library included identical crRNAs targeting genes encoding 45 total factors that were reported to be highly-expressed in H358 cells and play diverse roles such as chromatin remodeling (e.g., ACTL6A, CFID3, SMARCA4), histone modification (e.g, EHMT1, EHMT2, KAT6A, KDM1A), and DNA methylation (e.g., DNMT1, MECP2) (Fig. IB). A single vector was generated per gene, with each gene targeted by a pair of crRNAs. Based on prior experience with single vs. multiguide targeting using Cas9, it was possible that two unique crRNAs directed towards the same gene would increase tlie KO efficiency when co-expressed with enAsCas!2a.

[0145] Beginning with the CROP-seq format library, a 3’ 19bp barcode separated by a direct repeat and a barcode segment-specific primer site was appended to each pre-crRNA array to assist with both pre-crRN A capture and accurate single cell assignment of each crRNA pair (Figs 1C-1D). Sequencing amplicon preparation was optimized for either the full-length or barcode-only regions of the pre-crRNA cassetes (Fig. ID). To map the full-length pre-crRNA sequences, a computational method was developed based on creation of a custom pre- crRNA transcriptome with tire Cell Ranger algorithm, whereas mapping and assignment of cells to each barcode was performed using a standard scRNA-seq analysis pipeline (Fig, 5).

[0146] scRNA-seq of pre-crRNAs expressed from the direct-capture Perturb-seq format resulted in poor guide recovery, whether the cells contained enAsCasl2 and with different batches of encapsulating beads from 10X (Figs. 6A-6C).

[0147] The pre-crRNA length in the direct-capture Perturb-seq format was too short for proper detection. To address this, a direct-capture CROP-seq format was adopted, where capture sequences were appended to the pre-crRNA cassette in CROP-seq constructs. This permited guide RNA transcript complementarity to oligo(d)T or cognate capture sequences and amplification with either of two different reverse primers (Illumina vs Nextera sequences) during scRNA-seq library preparation. This separated RNAs bound through polyA:polyT interactions or from the capture sequences (see schema in Fig. 6D). Upon comparing tire two RNA capture methods for the same constructs, it was observed that sample preparation using the oligo(d)T-based approach was superior to the use of specific capture sequences (Figs. 6E-6F). I’he PCR amplification of full-length pre-crRNAs from direct-capture Perturb-seq constructs did not produce a discrete product of the expected size (Figs. 6B-6C) and this could be due to the template switch oligo (TSO) used as one of the primers, thereby leading to off-target amplification and reduced pre-crRNA detection. To address this, a unique primer binding sequence was added to the 5 ’ end of the pre-crRNA upstream of the first direct repeat. Upon expression in context, with the guide, this sequence would be cleaved off by enAsCasl2a during crRNA processing (Fig. 6G). Priming from this sequence resulted in a specific amplicon for the full-length pre-crRNA (Fig. 6H). However, the improved full-length pre-crRN A amplification quality did not enhance detection from direct-capture Perturb-seq format (Fig. 6H), which was again poor compared to the CROP-seq format used in a parallel scRNA-seq experiment (Fig. 61).

[0148] Although comparable results were achieved in targeting efficiency for multiple cell surface receptors in bulk for both CROP-seq and direct-capture Perturb-seq formats, the CROP-seq variant offers a streamlined approach through standard oligo(d)T-based enrichment with efficient and reliable detection of pre-crRNAs at the single-cell level.

[0149] Next, recovery of pre-crRNAs from cells transduced with the CROP-seq library’ was compared in the presence or absence of enAsCas!2a. Library transduction conditions were optimized for integration of ~1 vector per cellular genome, and this was followed by four days in culture after antibiotic selection to facilitate pre-crRNA expression and target gene disruption. The barcode sequencing strategy enabled relatively higher fraction assignment of pre-crRNAs to individual cells when compared to the full-length pre-crRNA approach (Figs.

[0150] 1E-1F). However, in cell populations expressing enAsCasl2a, substantially fewerpre-crRNAs and reduced consistency in assignment of pre-crRNAs within each cell was detected, regardless of the method for sequencing library preparation (Figs, lE-lG), To account for potential vector-specific influences on pre-crRNA recovery, a similar senes of screens and sequencing studies were performed with an otherwise equivalent direct-capture library. This format showed inadequate pre-crRNA recovery’ and was deprioritized for further assessment (Figs. 6A-6B). One possibility was that Casl2a’s self-processing activity was leading to degradation of pre-crRNA-containing transcripts and, as a result, sub-optimal assignment of perturbation sequences within individual cells.

[0151] Example 3: Efficient recovery of guide detection after degron -mediated removal of Casl2a

[0152] Elimination of enAsCasl2a from library-transduced cells following gene disruption i.e. triggered DNA damage) could provide an opportunity’ for pre-crRNA accumulation and improved recovery' in single cells. As a simple, pharmacological means to accomplish this, the degradation tag (dTag) system was leveraged to enable rapid and complete degradation of Casl2a. Within a standard lenti viral vector, an enAsCas 12a transgene was engineered to be expressed with a C-terminal FKBP12F36fusion domain (enAsCasl2a-FKBP12F36X) to enable selective ubiquitination and, subsequently, degradation of the protein upon addition of a dTag small molecule (e.g. dTagVl). After validating drug -induced degradation of the enAsCas 12a-FKBP12F36Vprotein in stably-expressing II358 cells (Fig. 7A-7B), the impact of this fusion on gene targeting efficiency in the presence or absence of dl'agV 1 was tested. As before, lentivinis-delivered pre-crRNAs were used to deplete CD81 and CD9 or CD47 and CD63, this time in cells expressing either enAsCas 12a or enAsCas 12a-FKBP12F36V(Fig. 2A). In the absence of dTagVl, enAsCas 12a and enAsCas 12a-FKBP12F36Vshowed similar levels of targeting efficiency (Fig. 2B). Continuous culture of enAsCasl2a-FKBP12F 6Xwith dTagVl (before and after pre-crRNA delivery) prevented knock-out for three out of four genes; only CD9 receptor levels were reduced in cells treated with dTagVl. This may be due to residual but undetectable enAsCas 12a combined with a highly -potent CZ)9-targeting spacer sequence / crRNA and / or potential differences in turnover or membrane trafficking kinetics of CD9 protein relative to the other targets that were evaluated.

[0153] After establishing that enAsCas 12a-FKBP12F36Vmaintained high gene editing capacity, the epigenetic factor screen was repeated with dTagVl -mediated depletion of enAsCas 12a-FKBP12F36Vfor 24 or 48 hours before collecting samples for single cell sequencing (Fig. 2C). Analysis of pre-crRNA counts showed continued improvement in guide recovery over the time points tested with dTagVl treatment (Figs. 2D-2E). For both the 24- and 48-hour treatments recovery and assignment pre-crRNAs to individual cells with frequencies similar to library-infected populations devoid of enAsCas!2a (up to 80%) was possible (Figs. IF, 2F). In line with a generalized negative influence from enAsCas 12a expression on pre-cRNA recovery, similar results w'ere observed with full-length pre-crRNA counts (Figs. 8A-8C) in addition to improved consistency when matching pre-crRNAs to individual cells with either the full-length or barcode only methods (Fig. 8D). Increased pre- crRNA recovery was observed following a 48-hour pulse with dTagVl in a separate cell line (K562, chronic myeloid leukemia) engineered to express CROP-seq-formatted pre-crRNAs and enAsCas!2a-FKBPl 2F36V(Fig. 9). These results demonstrated the utility' of decreasing enAsCas 12a levels immediately prior to Perturb-seq sample collection.

[0154] Expression profiles following disruption of lysine demethylase 1A (KDM1A / LSD1) and N -acetyltransferase 10 (NAT 10) exemplify both the specificity' and efficacy of phenotypes generated using the dual crRNA library (Figs. 2G-2H); a heatmap summarizing gene expression changes for alt perturbations can be found in Fig. 10A. All profiles were normalized to non-targeting control pre-crRNAs to account for dTagV1-induced effects on the transcriptome. KDM1A is known to directly inhibit expression of vimentin (VIM)., which represents the most upregulated mRNA in cells with the KDM1A pre-crRNA (Fig. 2G). It was also observed upregulation of individual genes within the HLA-I family (i.e. HLA-B, C, and E) in the same cells (Fig. 2G). It was recently shown that pharmacological targeting of KDM1A induced strong expression of several HLA-I genes in a panel of Small Cell Lung Cancer lines, with the increase in HLA-1 expression occurring only after seven or more days of culture in the presence of the inhibitor. While this coincides with die time point selected for the screen, it is possible that these (and other differences in gene expression observed in the screen) are secondary effects of target disruption, and distinct changes might be detected through an earlier time course analysis. Separately, consistent with its role in promoting ribosome biogenesis., 15 of the top 25 most down-regulated mRNAs in cells containing the NAT 10 pre-crRNA array encode for unique ribosomal subunits (Fig. 2H), which was also reflected in gene ontology analysis where 4 out of top 5 biological pathways were related to translation (Fig. 21).

[0155] By establishing a versatile platform comprising an enhanced, multiplexed pre-crRNA expression cassette with a chemically-degradable enAsCas12a variant, existing barriers to single cell guide RNA recovery and assignment for functional genomic screens that make use of this uniquely -capable DNA nuclease were overcome. A key challenge in adapting Casl2a to Perturb-seq had been the recovery of pre-crRNA-containing transcripts, necessary for linking individual guides to expression changes within associated, single cells post-gene targeting. This was addressed as described herein through acute degradation of Casl2a prior to RNA-seq library preparation, which prevented Casl2a-mediated cleavage of these pre- crRNA-containing transcripts and, consequently, reliable and robust pre-crRNA detection via scRNA-seq.

[0156] To support a range of experimental designs, optimized methods for library preparation enabling either full-length pre-crRNA sequencing or guide RNA identification via a pre- crRNA-linked barcode were developed. A proof-of-concept, loss-of-function screen targeting a subset of epigenetic factors demonstrated that the pre-crRNA expression vector library' and degradable enAsCas!2a system facilitated precise and efficient gene disruption.

[0157] While the approach described in the Examples herein employed the FKBP12F36domain and a corresponding dTag molecule for induced degradation of enAsCas!2a, emerging degron technologies, including direct targeting with a PROTAC molecule or a small molecule inhibitor of RNP formation, including the use of heparin,, could provide alternative options for speed and flexibility and more effective Casl2a reduction before sample collection for sequencing. These strategies could be especially useful in contexts where dTAG delivery or kinetics present limitations. Overall, the framework for the application of enAsCas12a-mediated Perturb-seq significantly amplifies the potential and broadens the opportunity space for this powerful genetic screening technology.

[0158] Example 4: Tuneable Casl2a-based CRISPR interference

[0159] To extend the capabilities of the Cas12a-degron system described herein beyond gene knockout, a CRISPR interference (CRISPRi) strategy designed for transcriptional silencing without genomic disruption was assessed. Accordingly, HyperLbCasl2a-ZIM3, a Lachnospiraceae Cas 12a variant fused to the KRAB-ZIM3 repressor domain, was engineered as described herein. This gene editor achieved robust, multiplexed repression of endogenous targets including CD81, CD47, and CD9 following delivery of a single pre-crRNA expression vector (Figs. 11A-11C). HyperLbCas12a-ZIM3 consistently outperformed existing Acidaminococcus Cas12a-based repressors in direct benchmark assays and across distinct cell types (Fig. 11C). Importantly, HyperLbCas12a-ZIM3 retained full functionality in the context of the herein-described degron system, thereby allowing tight control of Cas 12a levels via dTagVl treatment.

[0160] To determine feasibility of a Casl2a CRISPRi system, several known Cas 12a variants were generated including dead enAsCas12a (denAs, D908A), multiAsCas12a (multiAs), and dead HyperLbCas12a (HyperLb) (Figs. 11 A- 1 IB). AsCasl2a and LbCasl2a constructs used the same crRNA sequences but had different direct repeats (DR). No target gene expression inhibition was observed when HyperLb Cas12a variant was used with AsCas12a DRs under the conditions tested, and the reciprocal case was also true (data not shown).

[0161] The HyperLb Cas 12a variant showed the highest efficiency in repressing the three target genes in two cell lines (Fig. 11C). The dead enAs Casl2a construct showed only- minimal gene expression inhibition, further emphasizing the necessity of Cas 12a protein sequence optimization performed for the two other variants. MultiAs Cas 12a induced lower target knock-down than HyperLb (with the same crRNA sequences) and there was evidence of MultiAs Cas 12a nickase activity affecting target DNA integrity after prolonged expression of the Cas 12a system (14 days). After target gene repression had been established with HyperLb Cas12a, a portion of cells were treated with dTagV1 to degrade the CRISPRi system and target gene expression was monitored via flow cytometry (protein level) and qPCR (RNA level) (Fig.

[0162] 1 ID). Some depression was observed of one (CD47) out of three targets at either RNA or protein level after 24h of dTagVl treatment (Figs. 1 IE-1 IF). By the 48h time point, all three targets showed some level of gene expression activation,

[0163] Besides differences between three targets, variability was observed in gene expression changes at RNA and protein levels that were cell type specific. Protein levels of both CD81 and CD47 targets were easier to clear in DLD-1 than K562 cells (Fig. 11C). For CD47, 24h treatment with dTagVl increased target RNA expression from -12.5% to just under -50% of non-targeting control values in both K562 and DLD-1 cells, but at the protein level only K562 showed changes, with CD47 protein in DLD-1 cells only starting to appear after 72h of dTagVl treatment.

[0164] Unlike Casl2a-mediated knockout, where edits are permanent, CRISPRi requires sustained presence of the repressor to maintain gene silencing. Upon dTagVl -induced degradation of HyperLbCasl2a-ZIM3, rapid target re-expression was observed., with silencing began to reverse within 24 hours and was largely lost by 48 hours under the conditions tested (Figs. 1 ID-1 IF). This presented an unexpected limitation for Perturb-seq, where Casl2a must be cleared just before scRNA-seq to preserve RNA integrity. Without being bound to any particular theory, it is believed that loss of transcriptional repression leads to reactivation of target genes and corresponding transcriptomic shifts, which led to decoupling of the observed expression profiles from the intended perturbations.

[0165] To overcome this limitation, Casl2a was fused to epigenetic effectors, such as DNA methyltransferases, to produce CRISPRoff-like constructs. While conceptually promising, constitutive expression of these CRISPRoff-like editor proved incompatible in our cell models under the conditions tested. Inducible or next-generation systems such as CHARM may provide a path forward for durable Casl2a-mediated repression compatible with degron-based transcriptomic screens.

[0166] Despite its poor performance in Perturb-seq under conditions, HyperLbCasl2a-ZIM3 enabled strong, tunable, and multiplexed gene repression, which may make it suitable for contexts where acute degradation is not required prior to readout, thereby expanding the functional scope of Cas12a.

[0167] The HyperLbCas12a-ZIM3, a compact and potent transcriptional repressor that expanded the capabilities of Casl2a beyond genome editing supported strong and multiplexed gene silencing from a single guide array, making it well suited for multi -gene targeting or compact repression screens. Despite its potent target silencing effects, the transient nature of CRISPRi, powered here by the KRAB-ZIM3 domain, limited the HyperLbCasl2a-degron -based approach’s compatibility with Perturb-seq under the conditions tested. However, it is believed that these were really a challenge of timing and stability, not necessarily of incompatibility. Future adaptations that incorporate more persistent repression mechanisms e.g. low-toxicity, high-efficacy epigenetic silencers) or refined temporal control may bridge this gap. Regardless, the compositions described herein could be useful in screening applications where temporary repression of a primary transcriptional regulator is enough to elicit sustained secondary transcriptional changes. Upon degradation of the CRISPRi machinery, despite re-expression of the primary target gene, one would be able to obtain scRNA seq readout of the secondary changes with accurate primary target sgRNA assignment.

[0168] Together, the enAsCas12a-degron and HyperLbCas12a-ZIM3 repressor systems described herein expand the toolkit available for CRISPR-based functional genomics, enabling more compact, controllable, and scalable gene perturbation strategies, all within a framework that can evolve as next-generation effector domains and inducible control mechanisms become available. The ability to effectively employ Casl2a for gene knockout combined with Cas9 for CR1SPR activation has recently been demonstrated. The enAsCas12a-degron system described herein would now allow for efficient orthogonal gene perturbation experiments with a single cell RNAseq readout. Together, these capabilities could be critical for advancing both basic discovery and applied efforts in disease modeling, therapeutic target validation, synthetic biology, and large-scale screening efforts.

[0169] Methods

[0170] Cell culture

[0171] Parental NC1-H358 cell line (an epithelial-like cell that was isolated from the bronchiole of a male patient with bronchioalveolar carcinoma) was maintained under mycoplasma-free conditions. Cells were cultured in RPMI-1640 growth medium -<■ 10% heat inactivated FBS (Sigma, F4135) + 2mM L-Glutamine and 100 U / mL Penicillin-streptomycin (Gibco, 15140- 122).

[0172] Lentiviral production and cell transduction Plasmids expressing desired cargo (enAsCas12a or pre-crRNA) and lentiviral packaging constructs (VSVg and Delta8.9) were transiently co-transfected into HEK293T cells with Lipofectamine 2000. After 48 hours, supernatants were collected, filtered through a 0.45μM PES filter and concentrated with Lenti-X Concentrator (Takara, 631232) according to the manufacturer’s manual.

[0173] Transduction was performed in 8 pg / ml polybrene (Sigma Cat#TR-1003r) with direct addition of filtered and concentrated viral supernatant. Transduced cells were selected with 15 pg / ml blasticidin (for enAsCas 12a constructs) or 1 pg / ml puromycin (for pre-crRNA constructs and libraries). MOI of lentiviral infection was assessed with CellTiter-Glo® Luminescent Cell Viability Assay (Promega, G7572).

[0174] Vector Sequences

[0175] Transgene sequences for enAsCas12a, enAsCas12a-FKBP12F36Vdead enAsCas12a, multiAsCas12a, HyperLb Cas12a and pre-crRNA sequences are detailed in Tables 11-23 below, crRNA sequences for CD47 and CD63 were sourced from the literature. Knock-out crRNA sequences for CD81, CD9, epigenetic factors, and non-targeting controls (NTCs) and CRISPRi crRNAs (targeting CD81, CD47, and CD9) were designed using crisprVerse Bioconductor ecosystem using the GRCh38 genome build. Sequences not found in the human genome or targeting olfactory receptors were used as non-targeting controls (NTCs) to account for non-cutting and cutting but non-targeting activity. Different direct repeats were used in the pre-crRNA containing multiple crRNAs to limit possible recombination. The same direct repeat was used for Lb Casl2a pre-crRNA. pre-crRN A sequences were produced as DNA oligonucleotides and cloned into a puromycin-selectable lentiviral vector pLKO_SHC201 (Sigma) by Genscnpt™. For libraries, final plasmids were pooled together in equimolar ratios, concentrated (Millipore, UFC5010), and column purified (Qiagen™, 28004).

[0176] Establishment of CRISPRi cell lines

[0177] Stable cell lines were generated by electroporation of piggyBac transposon (PB) and transposase (PBO, Transposagen / Hera BioLabs) plasmids with SF Cell Line Kit (Lonza, V4XC-2032) using a 3:1 PB:PBO ratio (1 μg total DNA) into 1×106cells in 20pl reaction. FF-120 code was used for K562 cells and CM-130 for DLD-1 Expression of Casl2a constructs was confirmed with mScarlet fluorescence and selected with 10 pg / ml blasticidin treatment.

[0178] Flow cytometry

[0179] For surface protein analysis, staining was performed using the following antibodies: anti-human CD47 (FITC, Cat No. 323106), anti-human CD63 (PE-Cy7, Cat No, 353010), anti-human CD81 (APC, Cat No. 17-0819-42), and anti-human CD9 (PE, Cat No. 312106). Each antibody was used at a 1:25 dilution. The staining was conducted for ~60 minutes on ice in the MACS® buffer. Then, cells were washed twice with the MACS® buffer and fixed with 1% PFA for 5 minutes at RT to inactivate any remaining lentiviral particles, followed by a final wash with the MACS® buffer. Flow cytometry was performed on a BD FACSymphony instrument and data was analyzed using FlowJo software, plotted in BioRender™ and statistical analysis performed with R 4.0.0.

[0180] Single-cell RNA sequencing

[0181] Plasmid libraries consisting of 50 dual pre-crRNAs each (45 with two crRNA on one pre-crRNA targeting the same epigenetic factor and 5 NTCs) were transduced into H358-enAsCas12a or H358-enAsCas12a-FKBP12F36V cells at targeted MOI of 0.3 and a minimum coverage of 1000x cells per pre-crRNA. 1ng / μl puromycin selection was initiated 48h posttransduction. Some cells were split before puromycin selection to confirm MOI with CellTiter-Glo® (Promega™, G7570). The confirmed MOI was 0.27 for the CROP-seq library and 0.38 for the direct-capture Perturb-seq library in the H358-enAsCas12a cells; and 0.29 forthe CROP-seq library in H358-enAsCasl2a-FKBP12F36V cells.

[0182] Cel! processing and library preparation

[0183] In the case of H358-enAsCasl2a cells, after the introduction of the targeting library and initiation of the puromycin, cells were cultured for a week, then dissociated, washed in IxPBS, counted to ensure high viability (>90%) and resuspended in 1% BSA in 1xPBS to process through the 10x Chromium Controller according to the manufacturer’s protocol (CG000316 or CG000418) with a recovery aim of 10,000 - 20,000 cells per lane. In the case of H358-enAsCasl2a-FKBP12F36V, priorto lOx processing, cells were cultured in 1μM dTAGV-1 (Tocris Bioscience, 6914) for 24 hours or 48 hours. dTagVl was continuously supplied until the step of reverse transcription. Gene expression libraries were prepared according to the lOx protocol.

[0184] Pre-crRNA library preparation

[0185] The CROP-seq and polyadenylated sequences from direct-capture CROP-seq constructs were amplified from cDNA with forward primer 5’-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTtggaaaggacgaggtacc (for full-length pre-crRNA) OR 5 -GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC'TTCCCAGAGCCACCGTTACAC (for barcodes) and reverse primer 5’- CTACACGACGCTCTTCCGATCT with the following conditions: 250ng cDNA, IpM final primer concentration in a 50pl reaction with NEBNext® Q5 Hot Start HiFi PCR Master Mix (NEB, M0543L) cycling at 98°C - 3’, (98°C - 30". 66°C - 10”, 72°C - 30”) xll, 72°C - 1 lOpl from the finished PCR were used to add multiplexing indexes with 10μl of pre-mixed primers from Dual Index TT Set A (10x Genomics, PN-1000215) in a 50pl reaction with NEBNext® Q5 Hot Start HiFi PCR Master Mix (NEB, M0543L) cycling at 98°C - 3’, (98°C - 30”, 68°C - 10”, 72°C - 30”) x9, 72°C -1’. The long direct-capture CROP-seq transcripts bound to beads through capture sequences were amplified from cDNA with forward primer 5’- GTGACTGGAGTPCAGACGTGTGCTCTrCCGATCTtggaaaggacgaggtacc (for full-length pre-crRNA) and reverse primer 5’- GCAGCGTCAGATGTGTATAAGAGACAG with the same conditions as the polyadenylated transcripts amplification, except for the indexing primers, which were taken from Dual Index NT Set A (lOx Genomics, PN-1000242).

[0186] CRISPR libraries for direct-capture Perturb-seq and direct-capture CROP-seq constructs were created according to lOx genomics user manuals (CG000316 or CG000418).

[0187] Sequencing

[0188] Gene expression and pre-crRNA libraries were multiplexed and sequenced on NextSeq 2000. The intended read coverage for gene expression libraries was 20,000-30,000 reads per cell and a quarter of that for the pre-crRNA libraries. The gene expression libraries and pre-crRNA barcode libraries were sequenced with P3 kit at Rl: 28bp, R2: 90bp, and lObp each for i7 and i5. The full -length pre-crRNA libraries were sequenced with PI or P2 kits at varying length of R2 to cover the full-length of the pre-crRNA.

[0189] Data analysis Read alignment, demultiplexing and data pre-processing

[0190] Data analysis workflow is described in Figs. 6A-6I. Briefly, scRNA-seq reads were mapped to the reference human genome GRCh38-2020-A or a custom reference containing full length pre-crRNA sequences using Cell Ranger v7.1.0. To call 19bp barcodes from the reads kallisto-bustools were used. Demultiplexing of pre-crRNA feature barcoding data and assigning pre-crRNA to cells was performed with a custom script as described in Xie et al., 2019. This analysis was performed for both full length pre-crRNA sequences or barcodes alone. Downstream analysis was conducted using the assigned gene expression and pre- crRNA or barcode matrix. Cells with either more than 10% mitochondrial reads or less than 3,000 UMIs were removed. Cells assigned to a perturbation with less than 15 total assigned cells were removed. Genes with less than 500 UMIs across all cells were removed, leaving a total of 12,522 genes. Single cell expression matrix and feature barcodes were processed in Scanpy. Expression counts per cell were normalized to a total of 10,000 counts per cell, and normalized values were log transformed (natural log), after adding a pseudo-count of 1.

[0191] Statistical Analysis of transcriptome-wide changes in Epigenetic factor screen data A linear model (ordinary least-square regression) was fit for each of the 12,522 (response) genes, where the gene expression values were the response variable and the perturbation (using the NTC as a reference) as well as tire total number of UMIs were used as covariates. Statistical significance of the coefficient linking the perturbation identifier to the response gene expression was tested. The false discovery rate was controlled using the Benjamini-Hochberg procedure across all the tested genes.

[0192] Western Blot

[0193] enAsCas12a and enAsCas12a-FKBP12F36V-expressing NCI-H358 cells were treated with 1 μM dTAGV-1 (Tocris Bioscience, 6914) for various time points (48 hours, 24 hours, 6 hours, 2 hours, 30 minutes, and 10 minutes) or with no dTAGV-1 compound as a control (DMSO alone). Following treatment, cells were collected and lysed using RIPA Lysis and Extraction Buffer (Thermo Fisher™, 89900) supplemented with complete, EDTA-free Protease Inhibitor (Roche™, 11873580001) according to the manufacturer's instructions. Protein samples were prepared with 4x Laemmli Sample Buffer (Bio-Rad™, 16107472) containing 2 -Mercaptoethanol (Sigma, 63689) as per the manufacturer's instructions. The samples were then separated on Novex 4-12% Tris-glycine gels (Invitrogen, XP04122BOX) using Novex™ Tris-Glycine SDS Running Buffer (I0X) (Thermo Fisher™, LC2675) with Chameleon® Duo Pre-stained Protein Ladder (Licor, 928-60000) as the marker. Gels were transferred to PVDF membranes (Invitrolon™ PVDF / Filter Paper Sandwiches, 0.45 pm, 8.3 x 7.3 cm, LC2005) using Tris-Glycine Transfer Buffer (25X) (Thermo Fisher, LC3675). The membranes were then blocked with Intercept® (PBS) Blocking Buffer (Licor, 927-70001) for 2 hours at room temperature. Primary antibodies were incubated overnight at 4°C: Cas12a antibody [3D3-F7] (abeam, ab273438) at a 1: 1000 dilution and p- Actin Mouse Monoclonal Antibody for normalization (Licor, 926-42212) ata 1:2000 dilution. Following overnight primary' antibody incubation, membranes were washed with PBSt and incubated with IRDye® 680RD Donkey anti-Mouse IgG Secondary' Antibody (Licor, 926-68072) at a 1:5000 dilution for 1 hour at room temperature. Membranes were again washed in PBS and protein bands were visualized using the Bio-Rad ChemiDoc MP Imaging System.

[0194] Table 1: FACS Data

[0195] CD47-FITC CD47-FITC CD63-PECy7 CD63-PECy7 Guide MFI %+ MFI %+

[0196] Guide 025 99.10743802 99.4 101.6485396 99.7

[0197] Guide 025 100.892562 99.4 98.35146043 99.1

[0198] Guide 034 10.31404959 5.13 1.532252548 4.03

[0199] Guide 034 9.884297521 5.04 1.491210069 4.55

[0200] Guide 035 12 7.34 1.792188248 4.3

[0201] Guide 035 11.63636364 8.15 1.655379985 4.33

[0202] Guide 036 12.66115702 8.21 1.997400643 4.45

[0203] Guide 036 12.42975207 8.89 1.956358164 5.34

[0204] Guide 037 12.46280992 8 2.257336343 15.5

[0205] Guide 037 11.07438017 6.2 1.97003899 14.3

[0206] Guide 038 12.26446281 7.25 5.458649702 39.1

[0207] Guide 038 9.32231405 5.05 3.789588891 40.5

[0208] Guide 039 75.9338843 96.6 78.48690061 99.1

[0209] Guide 039 79.33884298 97.4 82.29017033 99.3

[0210] Guide 040 77.25619835 96.4 70.26472399 98.8

[0211] Guide 040 90.18181818 98.6 86.10712087 99.4

[0212] Guide 041 69.52066116 91.3 85.36835625 99.3

[0213] Guide 041 65.81818182 90.7 74.57418428 98.8

[0214] Guide 042 44.95867769 63.4 89.92407141 98.8

[0215]

[0216] Guide 042 44.46280992 62.1 85.73773856 98.5

[0217] MFI = median fluorescence intensity

[0218] %+ = percent of positive cells (see Extended Data Fig. 1 for details on FACS gating Table 2: FACS Data

[0219] CD81-APC CD81-APC CD9-PE CD9-PE Guide MFI %+ MFI %+ Guide 025 99.60722702 98.7 99.79075518 98.2 Guide 025 100.392773 98.9 100.2092448 98.1 Guide 026 13.19717203 43.9 0.26821381 1.87 Guide 026 9.866457188 37.4 0.26821381 1.68 Guide 027 9.709347997 40 0.243484877 1.79 Guide 027 7.792615868 39.4 0.26821381 1.81 Guide 028 16.18224666 44.1 0.342400609 5.91 Guide 028 16.40219953 44.6 0.367129542 6.62 Guide 029 20.58130401 50.1 0.292942743 3.78 Guide 029 22.93794187 52.6 0.292942743 3.43 Guide 030 81.47682639 96.9 71.54270496 97.3 Guide 030 96.46504321 98.3 73.92048697 97.2 Guide 031 103.6606441 99 103.6903177 98.1 Guide 031 108.7824038 98.9 99.58151037 97.8 Guide 032 83.42498036 96.6 61.38482024 93 Guide 032 89.86645719 97.1 70.68670344 93.4 Guide 033 78.08326787 96 71.69488301 96.5

[0220]

[0221] Guide 033 75.85231736 95.1 74.22484307 96.7 MFI = median fluorescence intensity

[0222] %+ = percent of positive cells (see Extended Data Fig. 1 for details on FACS gating)

[0223] Table 3: Unpaired two-sample t-test with Welch’s correction (aka Welch’s t-test), p-value CD47-FITC CD47-FITC CD63-PECy7 CD63-PECy7 Condition1 Condition2 MFI %+ MFI %+ Guide 025 Guide 034 0.003931458 0.000303747 0.010642988 2.11E-05 Guide 025 Guide 035 0.004585146 0.002813042 0.01054489 0.00195592 Guide 025 Guide 036 0.005656444 0.002382495 0.010693499 9.93E-05 Guide 025 Guide 037 0.000251254 0.006207363 0.010145131 0.00060672 Guide 025 Guide 038 0.001210131 0.007509376 0.002172554 0.00192248 Guide 025 Guide 039 0.018479346 0.105136913 0.017006668 0.62483011 Guide 025 Guide 040 0.235200196 0.334095365 0.209800899 0.5527864 Guide 025 Guide 041 0.013802942 0.022726761 0.143314275 0.46732757

[0224]

[0225] Guide 025 Guide 042 0.005911429 0.011289481 0.049416635 0.19872739

[0226] Table 4: Unpaired two-sample t-test with Welch’s correction (aka Welch’s t-test), p-value CD81-APC CD81-APC CD9-PE CD9-PE Condition1 Condition2 MFI %+ MFI %+ Guide 025 Guide 026 0.008152555 0.035379624 0.001335674 2.56E-05 Guide 025 Guide 027 0.001840742 0.001144626 0.001283213 0.000187689 Guide 025 Guide 028 0.001405288 0.000657285 0.001284496 0.002009801 Guide 025 Guide 029 0.00431109 0.016096675 0.001336006 0.000462563 Guide 025 Guide 030 0.379414912 0.331699731 0.023529438 0.006116265 Guide 025 Guide 031 0.242123477 0.349885593 0.571294985 0.395870342 Guide 025 Guide 032 0.146183159 0.051281542 0.086092372 0.018361129

[0227]

[0228] Guide 025 Guide 033 0.016883436 0.075546951 0.025826619 0.015511229 Table 5: FACS Data

[0229] CD CD CD8

[0230] CD63- CD47- 47- 63- CD81 1- CD9- CD9

[0231] PECy

[0232] FITC FIT PE -APC AP PE -PE 7

[0233] C Cy7 C

[0234] Guid Guid Guid Guid

[0235] MFI %+ MFI %+ MFI %+ MFI %+ e e e e

[0236] Guid Guid Guid 97.55 Guid 131.9

[0237] 77.558 89.780

[0238] e_02 87.2 e_02 86 e_02 32754 99.2 e_02 93675 99.4 5 91664 38914

[0239] 5 5 5 5 3

[0240] Guid Guid Guid 103.4 Guid 80.84

[0241] 113.96 105.44

[0242] e__02 98.2 e_02 96.7 e_02 99079 99.5 e__02 00239 99.1 5 41224 21113

[0243] 5 5 2 5 5

[0244] Guid Guid Guid 98.94 Guid 87.16

[0245] 108.47 104.77

[0246] e__02 98 e_02 96.9 e_02 76453 99.3 e__02 63007 99.3 5 6961 74995

[0247] 5 5 6 5 9

[0248] Guid Guid Guid 11.78 Guid 0.126

[0249] 3.3872 6.2415

[0250] e 03 37.6 e 03 36.4 e 02 63720 49.4 e 02 18780 4.84 66972 71951

[0251] 4 4 6 1 6 8 Guid Guid Guid 10.18 Guid 0.151

[0252] 3.2500 6.5016

[0253] e 03 41.9 e 03 37.3 e 02 15311 48.3 e 02 97801 8.76 87935 37449

[0254] 4 4 6 8 6 7

[0255] Guid Guid Guid 11.31 Guid 0.126

[0256] 3.3872 5.8370

[0257] e 03 36.6 e 03 34.5 e 02 28124 49 e 02 18780 4.44 66972 25621

[0258] 4 4 6 2 6 8 Guid Guid Guid 9.629 Guid 0.692

[0259] 7.0488 0.9651

[0260] e_04 41.8 e_04 8.46 e_04 04498 45.4 e_04 34429 29.8 92015 31959

[0261] 5 6 5 8 6 9

[0262] Guid Guid Guid 8.576 Guid 0.595

[0263] 6.5001 0.9275

[0264] e_04 40.2 e_04 9.01 e_04 69034 43.9 e_04 63101 27.7 75871 66943

[0265] 5 6 5 5 6 6

[0266] Guid Guid Guid 8.918 Guid 0.696

[0267] 6.5001 0.9651

[0268] e_04 39.4 e_04 9.65 e_04 70560 43.9 e_04 94969 29.5 75871 31959

[0269] 5 6 5 4 6 4

[0270] Guid Guid Guid 97.65 Guid 105.9

[0271] 103.62 98.610

[0272] e__10 98.8 e_10 94.4 e_10 41840 98.7 e__10 54133 96.2 11699 88279

[0273] 9 9 9 8 9 8

[0274] Guid Guid Guid 97.44 Guid 80.87

[0275] 100.27 98.832

[0276] e_10 98.6 e_10 96.2 e_10 94583 98.8 e_10 49536 97.2 85515 81276

[0277] 9 9 9 3 9 6

[0278] Guid Guid Guid 104.8 Guid 113.1

[0279] 96.100 102.55

[0280] e_10 93.5 e_10 84.4 e_10 96357 93.2 e_10 70912 92.4 27855 63045

[0281] 9 9 9 6 9 6

[0282] Guid Guid Guid 24.46 Guid 0.278

[0283] 19.533

[0284] 35.7 5.3016

[0285] e_11 e 11 32.9 e_11 47274 61.4 e_11 05730 28.6 42618 60365

[0286] 0 0 0 6 0 6

[0287] Guid 0.22

[0288] 20.055 Guid 09493 Guid 8

[0289] 10.72 Guid

[0290] e_11 22 e 11 12.2 e_11 43.9 e_11 80144 14.6 71031 67089 25113

[0291]

[0292] 0 0 0 0 1 CD CD CD8

[0293] CD63- CD47- 47- 63- CD81 1- CD9- CD9

[0294] PECy

[0295] FITC FIT PE -APC AP PE -PE 7

[0296] C Cy7 C

[0297] Guid Guid Guid Guid 0.219

[0298] 11.594 1.2970 16.35

[0299] e 11 13.5 e 11 16.2 e 11 62.2 e 11 26804 16.2 70752 57373 2.4695

[0300] 0 0 0 0 7

[0301] Guid 97.10 Guid 89.89 Guid 99.492 Guid 97.437

[0302] 99.2 97 27614 99.3 89831 97.7 e 11 1 08246 e l 44088 e 11 e 11

[0303] 11

[0304] 1 3 1 4 Guid 102.9 Guid 108.6 Guid 98.685 Guid 101.48

[0305] 99.4 98 96831 99.6 20239 98.6 e 11 1 3899 e 11 76602 e 11 e 11

[0306] 1

[0307] 1 1 1 5 Guid 99.90 Guid 101.4 Guid 101.82 Guid 101.07

[0308] 993 97.8 e 11 04074 99.5 e 11 80777 978 e 11 1 25276 e 111 4899

[0309] 1 2 1 3

[0310] Guid Guid Guid 16.02 Guid 0.2.36

[0311] 10.576 0.9932

[0312] 12.1 5.82 53508 53.1 97792 9.33 e_11 63579 e 11 066 e_11 e 11 2 39

[0313] 2 2 4 2 9

[0314] Guid Guid Guid Guid 0.253

[0315] 11.024 0.9596 14.99

[0316] e 11 13 3 6.31 52.1 04422 11.1 79833 e 11 6979 e_11 e 11 2 1 32096

[0317] 2 9

[0318] Guid Guid Guid 16.73 Guid 0.261

[0319] 9.3217 0.9261

[0320] e 11 11 6,35 15527 53.8 07737 12.3 80699 e 11 3294 e_11 e 11 2 4

[0321] 2 4 9

[0322] Guid Guid Guid 104.8 Guid 99.64

[0323] 99.797 97.846

[0324] e 11 99.3 e 11 96 e_11 58379 99.2 e 11 82208 99.2 86312 77497

[0325] 3 3 3 2 3 1 Guid Guid Guid 102.5 Guid 103.0

[0326] 101.96 105.86

[0327] 99.6 97.9 57041 99.6 87681 99.3 e 11 36154 e 11 36932 e_11 e 11

[0328] 3 3 3 7 3 3

[0329] Guid Guid Guid 92.58 Guid 97.26

[0330] 98.238 96.289

[0331] 99.4 96.5 45790 99.1 40978 99 e_11

[0332] 52151 e 11 53 e_11 e_11

[0333] 187

[0334] 3 3 7 3 7

[0335] Guid Guid Guid 16.81 Guid

[0336] 9.8758 1.1477 0.2.43 e_11 11.6 7.17 74665 53.1 9.47 30205 e 11 45 e_11 e_11

[0337] 843 53944 4 4 4 6 4

[0338] 0.262 Guid Guid Guid Guid

[0339] 9.7892 1.1102 14.48

[0340] 11.8 5.79 50 76623 107 e_11 e 11 e_11 e_11 00116 56657 66247

[0341] 4 4 4 4 8

[0342] 16.72 0.258 Guid Guid Guid Guid

[0343] 9.7025 0.9631

[0344] 104 5.12 89535 52.7 49361 102 e_11 e 11 e_11 e_11 70026 83697

[0345] 4 4 4 8 4 6

[0346]

[0347] MFI = median fluorescence intensity

[0348] %+:::percent of positive cells (see Figs 3A-3E for details on FACS gating)

[0349]

[0350] Table 6: Unpaired two-sample t-test with Welch’s correction (aka Welch’s t-test), p-value CD4 CD4 CD6 CD6 CD CD

[0351] 7- 7- 3- 3- 81- 81- CD CD FIT FIT PEC PEC AP AP 9- 9- C C y7 y7 C C PE PE Co

[0352] ndit

[0353] ion MF MF 1 2 MFI %+ 1 2 MFI %+ 1 2 I %+ 1 2 I %+ Gu Gu Gu Gu Gu 0.0 Gu Gu 0.0 0.0 Gui ide 0.01 0.00 ide ide 0.00 0.00 ide ide 001 1.2 ide ide 250 002 de 0 3466 1183 0 0 2924 2697 0 0 794 0E- 0 0 219 068 025 34 024 065 25 34 35 534 25 26 39 05 25 26 03 3 Gu Gu Gu Gu Gu 0.0 Gu Gu 0.0 Gui ide 0.01 0.00 ide ide 0.00 0.00 ide ide 002 5.1 ide ide 252 6.6 de...o 4409 3447 0 0 2654 1681 0...o 725 9E- 0 0 776 4E- 025 46 197 009 25 46 489 841 25 45 86 05 25 46 33 05 Gu Gu Gu Gu Gu 0.0 0.0 Gu Gu 0.0 0.0 Gui ide 0.00 ide ide 0.00 ide ide 002 141 ide ide 094 015 de_1 2.82 5061 1 -1 9.30 1742 1 -1 113 210 -1 I 877 484 109 10 E-05 279 09 10 E-07 446 09 10 11 44 09 10 86 35 Gu Gu Gu Gu Gu 0.0 Gu Gu 0.0 Gui ide ide ide 0.00 ide ide 001 7.11 ide ide 029 2.0 de2.81 5.15 1 _1 0168 6.61 1 _1 482 E- _1 _1 770 71 - 11! 12 E-06 E-05 11 12 244 E-08 11 12 93 05 11 12 97 05 Gu Gu Gu Gu Gu 0.0 0.0 Gu Gu 0.0 Gui ide 0.00 ide ide 0.00 ide ide 014 003 ide ide 002 5.3 de_ _1 0139 1.21 _1 _1 0894 4.39 _1 _1 071 155 _1 1 870 2E-

[0354]

[0355] 113 14 056 E-05 13 14 405 E-08 13 14 23 62 13 14 07 06

[0356] Table 7: FACS Data

[0357] CD81-APC CD81-APC

[0358] Guide MFI %+ Guide

[0359] Guide 025 89.55974843 98.3 Guide 025

[0360] Guide 025 110.4402516 99.1 Guide 025

[0361] Guide 101 59.72746331 81.7 Guide 101

[0362] Guide 101 63.10272537 82.5 Guide 101

[0363] Guide 102 55.87002096 79.3 Guide 102

[0364] Guide 102 54.04612159 78.2 Guide 102

[0365] Guide 105 65.78616352 81.8 Guide 105

[0366] Guide 105 57.75681342 80.7 Guide 105

[0367] Guide 106 54.2557652 78.5 Guide 106

[0368] Guide 106 50.71278826 78.1 Guide 106

[0369] Guide 103 52.80922432 81.1 Guide 103

[0370] Guide 103 52.80922432 80.3 Guide 103

[0371] Guide 104 45.97484277 76.5 Guide 104

[0372] Guide 104 47.31656184 77.2 Guide 104

[0373] Guide 107 43.10272537 76.1 Guide 107

[0374] Guide 107 43.35429769 76 Guide 107

[0375]

[0376] Guide 108 40.48218029 74.2 Guide 108 CD81-APC CD81-APC

[0377] Guide MFI %+ Guide

[0378]

[0379] Guide 108 41 57232704 74.8 Guide 108 MFI = median fluorescence intensity

[0380] %+ -- percent of positive cells (see Extended Data Fig. 1 for details on FACS gating)

[0381] Table 8: Unpaired two-sample t-test with Welch’s correction (aka Welch’s t-test), p-value CD81-APC CD81-APC Condition 1 Condition 2 MFI %+

[0382] Guide 025 Guide 101 0.160490319 0.001159253 Guide 025 Guide 102 0.142680375 0.001870761 Guide 025 Guide 105 0.13510631 0.00238803 Guide 025 Guide 106 0.129352215 0.002701308 Guide 025 Guide 103 0.138609944 0.000986194 Guide 025 Guide 104 0.121787656 0.000657498 Guide 025 Guide 107 0.115737467 0.010052855

[0383]

[0384] Guide 025 Guide 108 0.11075286 0.000684401

[0385] Table 9: FACS Data

[0386] CD47- CD47- CD63- CD63- CD81- CD81- CD9- CD9- FITC FITC PECy7 PECy7 APC APC PE PE Condition Guide MFI %+ MFI %+ MFI %+ MFI %+

[0387] Guide 99.082 89.773 88.051 86.160 Cas12a 025 56881 80.9 11791 98.6 16123 96.1 19033 98 Guide 102.52 105.60 105.16 105.82 Casl2a 025 29358 83.3 33001 99.2 66106 97.9 07772 99.1

[0388] Guide 98.394 104.62 106.78 108.01 Cas 12a 025 49541 80.8 3582 99.1 22282 98 90325 99.3

[0389] Guide 19.403 14.747 16.711 0.7698 Cas12a 034 66972 17.2 33585 35.4 54493 44.2 65186 4.08

[0390] Guide 19.403 16.809 12.722 0.6842 Cas 12a 034 66972 15.6 90031 41.5 98889 40 18874 3.17

[0391] Guide 22.981 18.356 12.722 0.6233 Cas12a 034 65138 22.6 82365 46.4 98889 38.6 1483 3.08

[0392] Guide 100.45 91.826 98.969 95.699 Casl2aDeg 025 15457 60.9 38106 95.5 07216 95.3 75157 98.5

[0393] Guide 99.096 99.520 98.608 103.79 Casl2aDeg 025 90865 59.6 85682 96.2 24742 95 15533 97 Guide 100.45 108.65 102.42 100.50 Casl2aDeg 025 15457 60.9 27621 97.2 26804 95.1 86951 98.6

[0394] Guide 29.385 17.249 24.226 1.8100 Cas12aDeg 034 2032 19.4 15445 31 80412 49.4 08281 21.6

[0395] Guide 30.739 18.940 20.876 1.3681 Casl2aDeg 034 84022 20.1 24803 33.3 28866 46.7 53318 19.8

[0396] Guide 29.385 19.701 17.113 1.1853 Casl2aDeg 034 2032 20.4 24014 34.1 40206 43.5 77972 18.8 Cas12a_luM Guide 97.959 91.662 94.605 97.414

[0397]

[0398] dTagVl 025 18367 82.2 15485 98.9 50459 98 62984 98.3 CD47- CD47- CD63- CD63- CD81- CD81- CD9- CD9- FITC FITC PECy7 PECy7 APC APC PE PE Casl2a__luM Guide 100.68 105.14 102.09 107.40 dTagVl 025 02721 83.6 34759 99.3 17431 98.7 75708 98.9 Casl2a_luM Guide 101.36 103.19 103.30 95.177 dTagVl 025 05442 83.1 43692 99.2 27523 98.6 79935 99.6 Casl2a_luM Guide 25.306 27.937 17.504 0.5697 dTagVl 034 12245 21 19545 66.8 58716 49.8 9617 4.14 Cas12a luM Guide 20.952 23.226 15.302 0.5525 dTagVl' 034 38095 16.8 85436 57.3 75229 47.6 89782 3.7 Casl2a_luM Guide 20.068 23.064 13.724 0.5016 dTagVl 034 02721 15.8 4288 56.8 77064 46 32401 3.58 Casl2aDeg_ Guide 91.909 91.791 93.609 luMdTagVl 025 100 52.8 95427 90.9 49191 90.5 36094 95.1 Casl2aDeg_ Guide 96.836 100.03 95.326 110.72 luMdTagVl 025 98297 51 51741 91.8 54284 90.9 60726 97.2 Casl2aDeg Guide 103.16 108.05 112.88 95.664 luMdTagVl 025 3017 54.7 48716 94.1 19652 94 56646 96.1 Casl2aDeg Guide 81.265 99.507 101.43 8.4608 luMdTagVl 034 20681 42.8 56243 85.2 79868 88.7 46085 51.8 Cas12aDeg__ Guide 78.102 97.819 94.427 9.5409 luMdTagVl 034 18978 41.5 20507 84.1 80108 87.6 54095 52.8 Cas12aDeg_ Guide 74.939 85.156 79.089 6.8106

[0399]

[0400] luMdTagVl 034 17275 39 5248 81.8 27501 83.8 81068 49.4 MFI = median fluorescence intensity

[0401] %+ = percent of positive cells (see Extended Data Fig. 1 for details on FACS gating) Table 10: Unpaired two-sample t-test with Welch’s correction (aka Welch’s t-test), p-value CD47- CD47- CD63- CD63- CD81- CD81 CD9 CD9 FITC FITC PECy7 PECy7 APC -APC -PE -PE Candid Conditi

[0402] onl on2 MFI %+ MFI %+ MFI %+ MFI %+ Cas12a _ Cas12a 0.000 0.004 Guide 0 Guide 1.48E- 0.0002 0.0027 0.0029 0.00344 23930 8593 1.19 25 034 06 84546 57197 28748 1941 4 99 E-08

[0403] Cas 12a

[0404] Cas 12a Deg G 0.001 0.000 Deg Gu uide 03 4.11E- 7.86E- 0.0030 8.69E- 3.18E- 19707 5294 1.16 ide 025 4 08 07 02017 06 05 4 19 E-06

[0405] Cas 12a

[0406] Cas 12a luMd

[0407] luMdTa TagVl 0.000 0.001 gVI Gu Guide_ 1.08E- 0.0002 0.0010 0.0068 000022 30039 4254 4 72 ide 025 034 05 90602 92746 84995 9313 2 7 E-07 Cas 12a Cas 12a

[0408] Deg In Deg_lu

[0409] MdTag MdTag 0.055 0.002 VI Gui VI Gui 0.0010 0.0016 0.4197 0.0034 0.41909 76445 9164 1.88

[0410]

[0411] de 025 de 034 60562 21384 51929 76873 6406 8 98 E-05

[0412]

[0413] APPENDIX A Vectors

[0414] pLK()_4XNLS_en AsCas 12a tcacccgcccgctctcgtcatcactgaggtggagaagagcatgcgtgaggctccggtgcccgtcagtgggcagagcgcacatc gcccacagtccccgagaagttggggggaggggtcggcaattgaacggtgcctagagaaggtggcgcggggtaaactgggaa agtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttc gcaacgggtttgccgccagaacacaggtaagtgccgtgtgtggtcccgcgggcctggcctctttacgggttatggcccttgcgtg ccttgaatacttccacgcccctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagttcgaggcct tgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcttgggcgctggggccgccgcgtgcgaatctggtggcacc ttcgcgcctgtctcgctgctttcgataagtctctagccattta aatttttgatgacctgctggacgctttttttctggcaagatagtcttgt aaatgcgggccaagatctgcacactggtatltcggttttggggccgcgggcggcgacggggcccgtgcgtcccagcgcacatg ttcggcgaggcggggcctgcgagcgcggccaccgagaatcggacgggggtagtctcaagctggccggcctgctctggtgcct ggcctcgcgccgccgtgtatcgccccgccctggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagatggcc gcttcccggccctgctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaag gaaaagggcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctcgattagttctcag cttttggagtacgtcgtctttaggttggggggaggggttttatgcgatggagtttccccacactgagtgggtggagactgaagttag gccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggatcttggttcattctcaagcctcagacagtggttcaaa gtttttttcttccatttcaggtgtcgtgaccctagcgctaccttagaGA ATT C CTTCTT AC AGA AT C GGAT CCc gccaccCTCGAGgccaccATGGCCAAGAGAACCGCCGACXXjCTCTGAGTTCGAGAG CCCCAAGAAGAAGCGGAAAGTGGGCAGCGGCACACAGTTCGAGGGCTTTACC AAC TGTATCAGGTGTCCAAGACACTGAGATTTGAGCTGATCCCTCAG GCAA GACCCTGAGCACATCCAGGAGCAGGGCTTCATCGAGGAGGACAAGGCCCGCA ATGATCACTACAAGGAGCTGAAGCCAATCATCCI TCGGATCTACAAGACCTAT GCCGACCAGTGCCTGCAGCTGGTGCAGCTGGATTGGGAGACCTGTCCGCCGCC ATCGACTCTTATCGCAAGGAGAAGACAGAGGAGACCCGGAACGCCCTGATCG AGGAGCAGGCCACATATCGCAATGCCATCCACGACTACTTCATCGGCCGGACA GACAACCTGACCGATCKX^TCAATAAGAGCiCACGCCGAGATCTACAAGGCiCCT GTTCAAGGCCGAGCTGTTTAATGGCAAGGTGCTGAAGCAGCTGGGCACCGTG ACX ACAACCGAGCACGAGAACaXX T(K TGAGATCCT CGATAAGTTTACACC TACTTCTCTGGCTTTTATCGGAACAGAAAGAACGTGTTCAGCGCCGAGGATAT CTCCACAGCCATCCCACACACKiATCGTGCACiGACAACTTCCCCAACJTTTAACSG AGAATTGTCACATCTTCACAAGACTGATCACGCCGTGCCAAGCCTGAGGGAGC ACTTTGAGAACGTGAAGAAG< KX: ATCGGCATCTTCGTGAGCACCTCCATCGAG GAGGTGTTTTCTTTCCCCTTTTATAACCAGCTGCTGACACAGACCCAGATCGAC CTGTACAACA(K: T(K: TGGGAGGAATCTCCAGAGAGGCAGGAACCGAGAAGAT CAAGGGCCTGAACGAGGTGCTGAATCTGGCCATCCAGAAGAATGATGAGACA CJCCCACATCATCGCCTCCCTCX: ACACAGATTCATCCCCCTGTTAACKAGATCC TGTCTGATAGGAACACCCTGAGCTTCATCCTGGAGGAGTTTAAGTCCGACGAG GAAGTGAT(X AGTCTTTCTGCAAGTACAAGACACTCKTGAGGAACGAGAATGT GCTGGAGACCGCCGAGGCCCGTTTAACGAGCTGAATAGCATCGACCTGACAC ACATCTTCATCTCCCACAAGAAGCTGGAGACCATCAGCAGCGCCCTGTGCGAC CACTGGGATACACTGAGAAATGCCCTGTATGAGCGGAGAATCAGCGAGCTGA CGGCAAGATCACCAAGAGCGCCAAGGAGAAGGTGCAGAGGTCCCTGAACJCAC GAGGATATCAACCTGCAGGAGATCATCAGCGCCGCAGGCAAGGAGCTGTCCG AGGCCTTCAAGCAGAAG CCTCTGAGAT(: TGTCCACGCACACGCCGCCCTGG ATCAGCCACTGCCTACAACCCTGAAGAAGCAGGAGGAGAAGGAGATCCTGAA GACXX: AGCTGGACTCCCTGCTG(K}CCTGTACCACCTGCT(K}ACT(X}TTTGCCC} TGGATGAGTCTACGAGGTGGACCCCGAGTTCAGCGCCAGGCTGACCGGCATC AAGCTGGAGATGGAGCCCTCTCTGAGCTTCTACAACAAGGCCC KJAATTATGC CACCAAGAAGCCATACAGCGTGGAGAAGTTCAAGCTGAACTTTCGATGCCCAC ACTGGCAAGGGGATGGGACGTGAATAGGGAGAAGAACAATGGCGCCATCCTG TTTGTGAAGAACGGCCTGTACTATCTGGGCATCATGCCTAAGCAGAAGGGCCG CTATAAGGCCCTGTCCTTCGAGCCACAGAGAAGACCTCTGAGGGC'mGATAA GATGTACTATG CT CTTCCCTGATGCCGCCAAGATG TCCCAAAGTGCTCCA CCCAGCTGAAGGCCGTGACAGCCCACTTTCAGACCCACACAACCCCAATCCTG CTTCTAACAATTTCATCGAGCCCCTGGAGATCACAAAGGAGATCTACGACCTG AACAATCCTGAGAAGGAGC’CAAAGAAGTTTCAGACAGCCTATGCCAAGAAGA CCGGCGATCAGAAGGGCTACAGAGAGGCCCTGTGCAGTGGATCGACTTCACA AGGGATTTTCTGTCIAAGTATACCAAGACAACCAGCATCGATCTGrCTAGC’Cr GCGCCCTTCCTCTCAGTATAAGGACCTGGGCGAGTACTATGCCGAGCTGAATC CACTGCTGTACCACTCAGCTTCCAGCCKJATCGCCGAGAAGGAGATCATGGATG CCGTGGAGACCGGCAAGCTGTACCTGTTCCAGATCTATAACAAGGACTTTGCC AAGGGCCACCACGGCAAGCCCAATCTGCACACACTGTATTGGACCXIGCTGTTT AGCCCTGAGAACCTGGCCAAGACATCCATCAAGCTGAATGGCCAGGCCGAGC TGTTCTACCGCCCCAAGTCCX JATGAAGAGAATGGCCCAC XKJCTGGGCG GAAGATGCTGAACAAGAAGCTGAAGGATAGAAGACACCAATCCCCGACACCC TGTACCACiGAC^TGTACGACTATGTGAATCACAGACTGTCTCACGACCTGACiC GATGAGGCAAGGGCCCTGCTGCCTAACGTGATCACCAAGGAGGTGAGCCACG AGATCATAAGGATAGGCGCTTrACCTCXX CAAGTTCTTTTTCCACGTGCCX^A TCACACTGAACTATCAGGCCGCCAATTCCCCTTCTAAGTTCAACCAGCGCGTG AATG( TA(: TGAAG(3A(X AC(XX: GAGACCCCTATCAT(3(KATC }ATAG€K3G CGAGCGCAACCTGATCTATATCACAGTGATCGACTCTACCGGCAAGATCCTGG AGCAGCGGAGCCTGAATACCAT(X: AGCAGTTTGATTACCAGAAGAAGCTCK]A CAACCGGGAGAAGGAGAGAGGGCAGCAAGGCAGGCATGGTCTGTGGTGGGC AC: AATCAA(}GATCTGAA(X: ACK}GCTATCTC}ACK: AGGTCATCCACC}AGATCG TGGACCTGATGATCCACTACCAGGCCGTGGTGGTGCTGGAGAACCTGAATTTC GCTTTAAGAGCAAGAGGACCGGAATC(K AGAGAAGGCCGTGTACCAGCAGTT CGAGAAGATGCTGATCGATAAGCTGAATTGCCTGGTGCTGAAGGACTATCCTG CCC}A AAGTGCKK 3< KX}T(}CTC}AAC CATA( }CT(3ACA(}ACCAC}TTCACC TCTTTTGCCAAGATGGGCACCCAGAGCGGCTTCCTGTTTTACGTGCCTGCCCCA TATACAA(X: AAGATCGAT(X XTGA( GGCTTCGTGGA( X TTCGTGTGGAA GACCATCAAGATCACGAGTCCCGGAAGCACTTCCTGGAGGGCTTCGACTTTCT GCACTACGATGTGAAGACAGGCGACTTCATCCTGCACTTTAAGATGAACAGAA ATCTGTCCTTCCAGAGGGGACTGCCAGGCTTTATGCCTGCATGGATATCGTGTT CGAGAAGAACGAGACACAGTTTGACGCCAAGG< K ACCCCTTTCATCGCCCX3C AAGCGCATCGTGCCAGTGATCGAGAATCACCGGTTTACCGGCAGGTACCGCGA CCTGTATCCXXK: AACGACK: TGATGCCCTGCTCK3AGGAGAACK}GCATCGTGTT CAGAGATGGCTCTAACATCCTGCCTAAGCTGCTGGAGAATGACGATAGCCACG CCATCGACACX: ATCKJTGGCCCTGATCCCX: TCCGTCKTGCAGATGCXK}AACTCT AATCCGCCACAGGCGAGGACTATATCAACTCCCCCGTGCGCGATCTGAATGG CGTGTGCTT(XJACTCTCXIGTTTCA(IAACCCA(JA(JTCXK CATGGA( JCCGATCI CCAATGGCGCCTACCACATCGCCCTGAAGGGCCAGCGCTGCTGAATCACCTGA AGGAGTCCAAGGATCTGAAGCTGCAGAACXKSCATCTCTAATCAGGACTCKiCT GGCCTACATCCAGGAGCTGAGAAACGCTCCCAAGAAGAAGCGGAAAGTAGGC AG( ]CC K}CTCXXK 3CCKK AGC X1ACCCAAGAA(}AA(3CCK}AAAGTCGACK} ACCCCAAGAAGAAGCGGAAGGTGGGGCGCGCCGGCAGTGGTGAGGGCAGAG GAAGrCTCX: TAACATGCG<}TC}ACGTCGAGGA(}AATCCTGGCCCAAT(}GCCAG CCTCTGTCCCAGGAGGAGTCTACCCTGATCGAGAGGGCCACCGCCACAATCA ACAGCATCCCAATCTCCGAGGACTACTCTGTGCK: AAGCXK: GCCCTGACK: TCC GATGGACGCATCl CACCGGCGTGAACGrGATCACrn'ACAGGCGGCCCCTGC GCCGAGCTGGTGGTGCTGGGAACCGCAGCAGCAGCAGCAGCAGGCAATCTGA CATGTATCGTGGCCATCGGCAACGAGAATAGGGGCATCCTGTCCCCATGCGGC CGGTGTAGACGGTGCTGCTGGACCTGCACCCCGGCATCAAGGCCATCGTGAAG GACTCTGATGGAC AGCCAACAGCAGTGGGCATC AGGGAGC TGCTGCCT AGCG GCTACGTGTGGGAGGGCTGAacgcgttaagtcgacaatcaactctggattacaaaatttgtgaaagattgactg gtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttct cctccttgtataaatcctggttgctgtctcttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctga cgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcgga actcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcat cgtccttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcg gaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgg gccgcctccccgcgtcgacttaagaccaatgacttacaaggcagctgtagatcttagccatttttaaaagaaaaggggggactg gaagggctaattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatctgagcctgggag ctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtga ctctggtaactagagatccctcagacccttagtcagtgtggaaaatctctagcagtacgtatagtagttcatgtcatcttattattcagt atttataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttatgcagcttataatggttacaaataaagcaatagcatc acaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgctctagctatc ccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgc agaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctagggacgtacccaattcgc cctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcggactgggaaaaccctggcgttacccaacttaatcg ccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctg aatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgc cagcgccctagcgcccgctccttcgcttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggc tccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgcc ctgatagacggtttcgccctttgacgtggagtccacgttctttaatagtggactcttgttccaaactggacaacactcaaccctatc tcggtctattcttttgatttataagggattttgccgatttcggcctattggtaaaaaatgagctgatttaacaaaaatttaacgcgaatttt aacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaa tatgtatccgctcatgagacaataaccctgataaatcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgc ccttattcccttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggt gcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagtttcgccccgaagaacgttttccaatgatgag cacttttaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcaga atgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataac catgagtgataacactgcggccaactacttctgacaacgatcggaggaccgaaggagctaacccttttttgcacaacatggggg atcatgtaactcgccttgatcgtgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagc aatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcgg ataaagtgcaggaccacttctgcgctcggccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcg cggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagtatctacacgacggggagtcaggcaactatggat gaacgaaatagacagatcgctgagataggtgcctcactgataagcattggtaactgtcagaccaagtttactcatatatactttgatt gatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttc cactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaa aaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaacttttttccgaaggtaactggcttcagcagagcgcaga taccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatc ctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagc ggtcgggctgaacggggggtcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgag ctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgc acgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttgtgatg ctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggcctttgctcacat gtctttcctgcgttatcccctgatctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgacc gagcgcagcgagtcagtgagcgaggaagcggaagagcgccaatacgcaaaccgcctctccccgcgcgttggccgattcatta atgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggca ccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgacc atgatacgccaagcgcgcaattaaccctcactaaagggaacaaaagctggagctgcaagcttaatgtagtcttatgcaatactcttg tagtcttgcaacatggtaacgatgagttagcaacatgccttacaaggagagaaaaagcaccgtgcatgccgattggtggaagtaa ggtggtacgatcgtgccttattaggaaggcaacagacgggtctgacatggattggacgaaccatgaatgccgcattgcagagat attgtatttaagtgcctagctcgatacataaacgggtctctctggttagaccagatctgagcctgggagctctctggctaactaggga acccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatcc ctcagaccctttagtcagtgtggaaatctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaaccagaggagct ctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttg actagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaa ttggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaat cctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggatcagaagaactta gatcattatataatacagtagcaaccctctattgtgtgcatcaaaggataagataaaagacaccaaggaagctttagacaagataga ggaagagcaaaacaaaagtaagaccaccgcac agcaagcGGC CGCTGATCTTC AGACCTGGAGGAG GAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAA AATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAG AGAGAAAAAGAGCAGTCKJGAATAGGAGCTTTGTTCCTTGCKJTTCTTGGGAGC AGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGACGGTACAGGCCAGA CAATTATTGTCTGGTATACJTGCAGCAGCAGAACAATTTGTGACKSGCTATTCiAG GCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGC AAGAATCCTCJGCT(}TCX3AAAGATAC CTAAACK}ATCAACAGCTCCTCiGG<}ATT TGGGGTTGCTCTGGAAAACCATTTGCACCACTGCTGTGCCTTGGAATGCTAGT TCK3AGTAATAAATCTCTCK1AACAGATTTGGAATCACACGACCTCK1ATGGAGT GGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAT CGCAAAAC: CAGCAAGAAAA( AATGAACAAGAATTATTGGAATTA(}ATAAATG GGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAAT TATTCATAATGATA(3TAG<jACi<iCTTG<yTA(iGTTAAC AATAGTTTTT(jCT(3TAC TTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACC CACCTCCCAACC CC(}ACK}GGACCCGACA(3GCCCC}AAC}<3AATAC}AAGAA(}AA GGTGGAGAGAGAGCAGAGACAGATCCATTCGATTAGTGAACGGATCTCGACG GTATCGGTTAACTTI AAAAGAAAAGGLKJGGATTGCK KKSTACAXGTGCAGGG GAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATACAAA AAC / XAATTACAAAAATTCAAAATTTTATCCxATgagtaattcatacaaaaggactcgcccctgcctt ggggaatcccagggaccgtcgttaaactcccactaacgtagaacccagagatcgctgcgttcccgcccc pLKO_4XNLS_enAsCasl2a-FKBP12F36V icacccgcccgctctcgtcatcactgaggtggagaagagcatgcgtgaggctccggtgcccgtcagtgggcagagcgcacatc gcccacagtccccgagaagttggggggaggggtcggcaattgaacggtgcctagagaaggtggcgcggggtaaactgggaa agtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttc gcaacgggtttgccgccagaacacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgt gcctgaatacttccacgcccctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagttcgaggc cttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcttgggcgctggggccgccgcgtgcgaatctggtggca ccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaattttgatgacctgctggacgcttttttctggcaagatagtctt gtaaatgcgggccaagatctgcacactggtatttcggtttttggggccgcgggcggcgacggggcccgtgcgtcccagcgcac atgttcggcgaggcggggcctgcgagcgcggccaccgagaatcggacgggggtagtctcaagctggccggcctgctctggtg cctggcctcgcgccgccgtgtatcgccccgccctggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagatg gccgcttcccggccctgctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacaca aaggaaaagggcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctcgattagttct cagcttttggagtacgtcgtctttaggttggggggaggggttttatgcgatggagtttccccacactgagtgggtggagactgaagt taggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggatcttggttcattctcaagcctcagacagtggttc aaagtttttttcttccatttcaggtgtcgtgaccctagcgctaccttagaGAA'n'CCITCTTACAGAATCGGATC CcgccaccCTCGAGgccaccATGGCCAAGAGAACCGCCGACGGCTCTGAGTTCGAGA GCCCCAAGAAGAAGCGGAAAGTGGGCAGCGGCACACAGTTCGAGGGCTTTAC CAACCTGTATCAGGTGTCCAAGACACTGAGATTTGAGCTGATCCCTCAGGGCA AGACCCTGAGCACATCCAGGAGCAGGGCTTCATCGAGGAGGACAAGGCCCGC AATGATCACTACAAGGAGCTGAAGCCAATCATCGATCGGATCTACAAGACCT ATGCCGACCAGTGCCTGCAGCTGGTGCAGCTGGATTGGGAGACCTGTCCGCCG CCATCGACTCTTATCGCAAGGAGAAGACAGAGGAGACCCGGAACGCCCTGAT CGAGGAGCAGGCCACATATCGCAATGCCATCCACGACTACTTCATCGGCCGGA CAGACAACCTGACCGATGCCTCAATAAGAGGCACGCCGAGATCTACAAGGGC CTGTTCAAGGCCGAGCTGTTTAATCKJCAACJGTGCTGAAGC CJCTGCJGCACCGT GACCACAACCGAGCACGAGAACGCCCTGCTGAGATCCTTCGATAAGTTTACAC CTACTTCTCTGGCTTTTAT(X3GAACAGAAAGAA(X3TGTTCAG(X]CCGAGGATA TCTCCACAGCCATCCCACACAGGATCGTGCAGGACAACTTCCCCAAGTTTAAG GAGAATTGTCACATCTTCACAAGACTGATCACGCCGTGCCAAGCCTGAGGGA GCACTTTGAGAACGTGAAGAAGGCCATCGGCATCTTCGTGAGCACCTCCATCG ACJGAGGTGTTTTCTTTCCCCTTTTATAACCACJCTCJCTGACACAGACCCAGATC GACCTGTACAACAGCTGCTGGGAGGAATCTCCAGAGAGGCAGGAACCGAGAA GATCAAGGGCCTGAACGAGGTGCTGAATCTGCXXIATCCAGAAGAATGATGAG ACAGCCCACATCATCGCCTCCCTGCCACACAGATTCATCCCCCTGTTAAGCAG ATCCTGTCTGATAGGAACACCCTGAGCTTCATCCTGGAGGAGTTTAAGTCCGA CGAGGAAGTGATCCAGTCTTTCTGCAAGTACAAGACACTGCTGAGGAACGAG AATGTGCTGC}AGACCGCCGA( JCCCGTTTAACGAGCT(}AATA(}CATCGACCT GACACACATCTTCATCTCCCACAAGAAGCTGGAGACCATCAGCAGCGCCCTGT CK: C}ACCACTGCK}ATACACTGAGAAATGCCCTGTAT(3ACKX}GA(}AATCACK: GA GCTGACGGCAAGATCACCAAGAGCGCCAAGGAGAAGGTGCAGAGGTCCCTGA AGCA( AGGATATCAACCTGCAGGAGATCATCACK JCCGCAGGCAAGGAGCT GTCCGAGGCCTTCAAGCAGAAGACCTCCGAGATCCTGTCCACGCACACGCCGC CCTGGATCAGCCXXCTGCCTACAA(XX: TGAAGAA K AGGAGGAGAAGGAGATC CTGAAGAGCCAGCTGGACTCCCTGCTGGGCCTGTACCACCTGCTGGACTGGTT

[0415] T(XXX3TGGATGAGTCTA(XI CK3TGGACX: CXX}AGTTCAGCG(X AG< K TGACCG GCATCAAGCTGGAGATGGAGCCCTCTCTGAGCTTCTACAACAAGGCCCGGAAT TATGCCACCAAGAAGCCATACAGCGTGC}AGAAGTTCAACK: TGAACTTTCGATG CCCACACTGGCAAGGGGATGGGACGTGAATAGGGAGAAGAACAATGGCGCCA TCCTGTTTC? TGAAGAACG< K TC}TACTATCTG€ K ATCATGCCTAA(K AGAAG GGCCGCTATAAGGCCCTGTCCTTCGAGCCACAGAGAAGACCTCTGAGGGCTTT GATAAGATGTACTATGACTACTTCCCTGATGCCXXX: AAGATGAT(X: AAAGTG CTCCACCCAGCTGAAGGCCGTGACAGCCCACTTTCAGACCCACACAACCCCAA TCCTGCTTCTAACAATTTCATCGzkCiCCCCTGGAGATCACAAAGGAGATCTACXj ACCTGAACAATCCTGAGAAGGAGCCAAAGAAGTTTCAGACAGCCTATGCCAA GAAGA(X JGCGATCAGAAGG K TACAGAGACK CTOTGCAGTGGAT(XJACT TCACAAGGGATTTTCTGTCTAAGTATACCAAGACAACCAGCATCGATCTGTCT ACXXTCX? XKXX: TT(: TCTCAGTATAACK}A(: TGCXXXJAGTACTATCXXX}ACXT GAATCCACTGCTGTACCACTCAGCTTCCAGCGGATCGCCGAGAAGGAGATCAT CK}ATGCCGTG<}AGACCGGCAAGCTGTACCT(3TTCCAGATCTATAAC / \AGGACT TTGCCAAGGGCCACCACGGCAAGCCCAATCTGCACACACTGTATTGGACCGGC TGTTTAGCCCTGAGAACCTG< KX: AAGACATCCATCAA(K: TGAATG< X: AGCXX: GAGCTGTTCTACCGCCCCAAGTCCCGGATGAAGAGAATGGCCCACCGGCTGG GCGAGAAGATGCTGAACAAGAAGCTGAAGGATAGAAGACACCAATCCCCGAC ACCCTGTACCAGGAGCTGTACGACTATGTGAATCACAGACTGTCTCACGACCT GAGCGATGAGGCAAGGGCCCT GCI GCCTAACGTGATCACCAAGGAGGTGAGC CACGAGATCATAAGGATAGGCGCTTTACCTCCGACAAGTTCTTTTTCCACGTG CCCATCACACTGAACTATCAGGCCGCCAAI CCCC1 CTAAGTTCAACCAGCG CGTGAATGCCTACCTGAAGGAGCACCCCGAGACCCCTATCATGGCATCGATAG GGGCGAGCGCAACCTGATCTATATCACAGTGATCGACTCTACCGGCAAGATCC TGGAGCAGCGGAGCCTGAATACCATCCAGCAGTTTGATTACCAGAAGAAGCT GGACAACCGGGAGAAGGAGAGAGGGCAGCAAGGCAGGCATGGTCTGTGGTG GGCACAATCAAGGATCTGAAGCAGGGCTATCTGAGCCAGGTCATCCACGAGA TCGTGGACCTGATGA TCCACI ACCAGGCCGTGGTGGTGCTGGAGAACCTGAAT TTCGCTTTAAGAGCAAGAGGACCGGAATCGCAGAGAAGGCCGTGTACCAGCA GTTCGAGAAGATGCTGATCGATAAGCTGAATTGCCTGGTGCTGAAGGACTATC CTGCCGAGAAAGTGGGCGGCGTGCTGAACCCATACCGCTGACAGACCAGTTC ACX TCTTTTGCCAAGATGGGC CC AGACX JG€TTCCTGTTTTACGTCKX T XX CCATATACAAGCAAGATCGATCCCCTGACCGGCTTCGTGGACCCCTTCGTGTG GAAGACCXITCAAGATCACGAGTCCCGGAAGCACTTCCTGGAGGGCTTCGACTT TCTGCACTACGATGTGAAGACAGGCGACTTCATCCTGCACTTTAAGATGAACA GAAATCTGTCCTTCCXXGAGGGGACTGCCAGGCTTTATGCCTGCATGGATATCG TGTTCGAGAAGAACGAGACACAGTTTGACGCCAAGGGCACCCCTTTCATCGCC GGCAAGCGCATCGTG X^AGTGATCGAGAATCACCGGTTTACCGGCAGGTACC GCGACCTGTATCCCGCCAACGAGCTGATGCCCTGCTGGAGGAGAAGGGCATC GTGTTCAGA(JATG< JCTCTAACATCCTGCCTAA(}CT(JCT(}GAGAATGA(X}ATAG CCACGCCATCGACACCATGGTGGCCCTGATCCGCTCCGTGCTGCAGATGCGGA ACTCTAATCXXSCCACAGGCGAGGACTATATCAACTCCXXXXJTGCGCGATCTGA ATGGCGTGTGCTTCGACTCTCGGTTTCAGAACCCAGAGTGGCCCATGGACGCC GATCKX: AATGCXX}CCTA(X ACATCGCCCTGAAGCK}CCACKX}CTC CT(}AATCAC CTGAAGGAGTCCAAGGATCTGAAGCTGCAGAACGGCATCTCTAATCAGGACT GGCTG< KX: TACATCCAGGAGCTGAGAAACGCTCCCAAGAAGAAGCCI AAAGT AGGCAGCGCCGGCTCCGCCGCGGCAGCGGACCCAAGAAGAAGCGGAAAGTC GAGGACXXX: AAC}AAC AACK JGAAGGTGCXjGCC X}CCG< KX}GATCCCKX ggagt gcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccgacctgcgtggtgcactacaccgggatgc ttgaagatggaaagaaagttgattcctcccgggacagaaacaagccctttaagtttatgctaggcaagcaggaggtgatccgagg ctgggaagaaggggttgcccagatgagtgtgggtcagagagccaaactgactatatctccgattatgcctatggtgccactgggc acccaggcatcatcccaccacatgccactctcgtcttcgatgtggagcttctaaaactggaaGGCAGTGGTGAC jGC AGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGCCCAATGG CCAACX: CTCTGT(XX AG<}AGC}AGTCTACCCT(}AT(XJA(JAG< XSCCACC XX AC AATCAACAGCATCCAATCTCCGAGGACTACTCTGTGGCAAGCGCCGCCCTGAG CT(XX}ATG }ACGCATCTTCA(XXKiCGTC}AACGTC}TATCACTTTACACXXXKiCC CCTGCGCCGAGCTGGTGGTGCTGGGAACCGCAGCAGCAGCAGCAGAGGCAAT CTCrACATGTATCX}TGGCCATCCK}CAACX}AGAATAG< K3GCATCCTGT(XX CATG CGGCCGGTGTAGACAGGTGCTGCTGGACCTGCACCCCGGCATCAAGGCCATC GTGAAG< IACTCTCIATGGACAGCCAACGCA(IT(KJGCATCAGG(}AGCT(JCTGCC TAGCGGCTACGTGTGGGAGGGCTGAacgcgttaagtcgacaatcaacctctggattacaaaatttgtgaaa gatgactggtattcttaactatgttgctcctttacgcttgtggatacgctgctttaatgccttgtatcatgctattgctcccgtatggct ttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactg tgttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttgctttccccctccctattgcca cggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggg gaaatcatcgtcctttcctggctgctcgcctgtgtgccacctggattctgcgcgggacgtccttctgctacgtccctcggccctca atccagcggaccttccttcccgcggcctgctgcggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctc ccttgggccgcctccccgcgtcgactttaagaccaatgacttacaaggcagctgtagatcttagccacttttaaaagaaaagggg ggactggaagggctaattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatctgagccg ggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttg tgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtacgtatagtagttcatgtcatcttatta ttcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaactgtttattgcagcttataatggttacaaataaagcaata gcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttateatgtctggctct agctatcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaatttttttta tttatgcagaggccgaggcgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctagggacgtaccca attcgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaac ttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgategcccttcccaaagttgcgc agcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctac acttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcg ggggctccctttagggtccgatttagtgcttacggcacccgaccccaaaaaacttgattagggtgatggttcacgtagtgggcca tcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaac cctatctcggtctattctttlgatttataagggattttgccgatttcggcctatggttaaaaaatgagctgattaacaaaaatttaacgc gaatttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacat tcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccg tgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctgtgaaagtaaaagatgctgaagatcagt tgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatcctgagagttttcgccccgaagaacgttttccaatga tgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattc tcagaatgacttggttgagtactcaccagtccagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccata accatgagtgataacactgcggccaacttactctgacaacgatcggaggaccgaaggagctaaccgcttttgcacaacatggg ggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgta gaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcg gataaagttgcaggaccacttctgcgctcggcccttccggctggctggttattgctgataaatctggagccggtgagcgtgggtct cgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtattatctacacgacggggagtcaggcaactatgga tgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttag attgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgacca aatcccttaacgtgagttttcgt tccactgaggtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaa aaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcag ataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacaacctcgctctgctaatc ctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagc ggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtga gctatgagaaagcgccacgcttcccgaagggagaaaggcggacagtatccggtaagcggcagggtcggaacaggagagcgc acgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgattttgtgat gctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggcctttgctcac atgtttttcctgcgttatcccctgattctgtggataaccgtattaccgccttgagtgagctgataccgctcgccgcagccgaacgacc gagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcatt aatgcagctggcacgacaggttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtagtagctcactcattaggca ccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgacc atgattacgccaagcgcgcaattaaccctcactaaagggaacaaaagctggagctgcaagcttaatgtagtcttatgcaatactctt gtagtcttgcaacatggtaacgatgagtagcaacatgccttacaaggagagaaaaagcaccgtgcatgccgattggtggaagtaa ggtggtacgatcgtgccttattaggaaggcaacagacgggtctgacatggattggacgaaccactgaattgccgcattgcagaga tattgtatttaagtgcctagctcgatacataaacgggtctctctggttagaccagatctgagcctgggagctctctggctaacaggga acccactgctaagcctcaataaagcttgcctgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatcc ctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaaccagaggagc tctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgagggcggcgactggtgagtacgccaaaaattttga ctagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaatt cggtaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatc ctggcctgttagaaacacagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggatcagaagaacttaga tcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagatagag gaagagcaaaacaaaagtaagaccaccgcacagcaagcGGCCGCT GAT CTTC AGAC CT GGAGGA G GAGATTGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAA ATTGAACCATTAGGAGT GCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGA GAGAAAAAAGAGC AGTGGGAA TAGGAGCITTGI’I'CCTTGGGTTCT TGGGAGC AGCAGGAAGCACTATGGGCG GCGTCAATGACGCTGACGGTACAGGCCAGAC AATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAG GCGCAACAGCATCTGTTGC ACTCACAGTCTGGGGCATCA GCAGCTCCAGCA AGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTG GGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTT GGAGTAATAAA TCTCTGGAACAGATTTGGATCAC ACGACCTGGATGGAGTGG GACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTT ATTGAAGAATC GCAAAACCA(K: AAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGG GCAAGTTTGTGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAAT A TTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTT TCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCTTTCAGACCCAC CTCCCAACCCCGACKiGGACX GACAG iCCCGAACKjAATAGAAGAAGAAGGT GGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTCGACGGT ATCGGTTAACTTTTAAAAGAAAACKjGGGATTG(KKiGGTACA(}TGCAG(}G<yAA AGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAAC AAATTACAAAAATTCAAAATTTTATCGATgagtaattcatacaaaaggactcgccctgccttggggaa tcccagggaccgtcgttaaactcccactaacgtagaacccagagatcgctgcgttcccgccccc pre-crRNA for cell surface receptors

[0416] Direct_

[0417] pre-crRNA Capture

[0418] Target(-s) Genome repeat- crRNAI ID Sequence

[0419] SI CTGAA

[0420] taatttct GGTGT

[0421] NTC,

[0422] Guide_#025 None hg38 actcttgt CTGGC

[0423] 0R1A2

[0424] agat AGAGC

[0425] TTA CAAGG

[0426] taatttct ACACC

[0427] CD9,

[0428] Guide..#026 None hg38 actcttgt TACAA

[0429] CD81

[0430] agat CAAGG

[0431] TGA CAAGG

[0432] taatttct ACACC

[0433] CD9,

[0434] Guide_#027 None hg38 actcttgt TACAA

[0435] CD81

[0436] agat CAAGG

[0437] TGA CAAGG

[0438] taatttct ACACC

[0439]

[0440] CD9,

[0441] Guide_#028 CS1. CS2 hg38 actcttgt TACAA

[0442] CD81

[0443] agat CAAGG

[0444] TGA CAAGG

[0445] taatttct ACACC

[0446] CD9,

[0447] Guide_#029 CS2. CS1 hg38 actcttgt TACAA

[0448] CD81

[0449] agat CAAGG

[0450] TGA CAAGG

[0451] taatttct ACACC

[0452] CD9,

[0453] Guide_#030 CS1. CS2 hg38 actcttgt TACAA

[0454] CD81

[0455] agat CAAGG

[0456] TGA CAAGG

[0457] taatttct ACACC

[0458] CD9,

[0459] Guide_#031 CS2. CS1 hg38 actcttgt TACAA

[0460] CD81

[0461] agat CAAGG

[0462] TGA

[0463]

[0464] Direct_ Direct_ Direct- Direct- repeat# crRNA2 repeat. crRNA3 repeat.. crRNA4 repeat- 2 #3 #4 #5 CCCCA

[0465] taatttct TCACA actGtC CTGAC None None None None None gtagat TGGAA ATC CCTGT

[0466] taatttct GAGGT actGtC GGCCG None None None None None gtagat CCGGC ATC CCTGT

[0467] taatttct GAGGT aatttcta actGtC GGCCG ctctAgt None None None None gtagat CCGGC agat ATC CCTGT

[0468] taatttct GAGGT actGtC GGCCG None None None None None gtagat CCGGC ATC CCTGT

[0469] taatttct GAGGT actGtC GGCCG None None None None None gtagat CCGGC ATC CCTGT

[0470] taatttct GAGGT actGtC GGCCG None None None None None gtagat CCGGC ATC CCTGT

[0471] taatttct GAGGT actGtC GGCCG None None None None None gtagat CCGGC ATC

[0472] V

[0473]

[0474] Full pre-crRNA sequence Barcode

[0475] gtaatttctactcttgtagatCT GAAGGT GT CT GGCAG AGCTTAtaatttctactGtCgtagatCCCCATCACAC None TGACTGGAAATCtttttt

[0476] gtaatttetactcttgtagatCAAGGACACCTACAACAA GCTGAtaatttctactGtCgtagatCCTGTGAGGTGG None CCGCCGGCATCttttt

[0477] gtaatttctactcttgtagatCAAGGACACCTACAACAA GCTGAtaatttctactGtCgtagatCCTGTGAGGTGG None CCGCCGGCATCAaatttctactctAgtagattttttt

[0478] cP> « gtaatttctactcttgtagatCAAGGACACCTACAACAA GCTGAGCTTTAAGGCCGGTCCTAGCAAtaattt

[0479] None ctactGtCgtagatCCTGTGAGGTGGCCGCCGGC ATCGCTCACCTATTAGCGGCTAAGGtttttt

[0480] gtaatttetactcttgtagatCAAGGACACCTACAACA AGCTGAGCTCACCTATTAGCGGCTAAGGtaa

[0481] None tttctactGtCgtagatCCTGTGAGGTGGCCGCCG GCATCGCTTTAAGGCCGGTCCTAGCAAtttttt

[0482] gtaattctactcttgtagatGCTTTAAGGCCGGTCCT AGCAACAAGGACACCTACAACAAGCTGAtaa

[0483] None tttctactGtCgtagatGCTCACCTATTAGCGGCTA AGGCCTGTGAGGTGGCCGCCGGCATCtttttt

[0484] gtaatttctactcttgtagatGCTCACCTATTAGCGGCT AAGGCAAGGACACCTACAACAAGCTGAtaattt

[0485] None ctactGtCgtagatGCTTTAAGGCCGGTCCTAGC AACCTGTGAGGTGGCCGCCGGCATCtttttt

[0486] . S'.

[0487]

[0488] CAAGG

[0489] taattict ACACC

[0490] CD9,

[0491] Guide_#032 CS1. CS2 hg38 actcttgt TACAA

[0492] CD81

[0493] agat CAAGG

[0494] TGA CAAGG

[0495] taattict ACACC

[0496] CD9,

[0497] Guide_#033 CS2. CS1 hg38 actcttgt TACAA

[0498] CD81

[0499] agat CAAGG

[0500] TGA ATACC

[0501] taattict AAACT

[0502] CD47,

[0503] Guide_#034 None hg38 actcttgt GTCCC

[0504] CD63

[0505] agat CAGAA

[0506] CAG TCCCA

[0507] taattict ATCTG

[0508] CD63, Guide.#035 None hg38 actcttgt TGTAG CD47 agat TTAGC

[0509]

[0510] AGC ATACC

[0511] taattict AAACT

[0512] CD47,

[0513] Guide_#036 None hg38 actcttgt GTCCC

[0514] CD63 agat CAGAA

[0515] CAG ATACC

[0516] taaittct AAACT

[0517] CD47,

[0518] Guide_#037 CS1, CS2 hg38 actcttgt GTCCC

[0519] CD63

[0520] agat CAGAA

[0521] CAG ATACC

[0522] taatttct AAACT

[0523] CD47,

[0524] Guide. #038 CS2. CS1 hg38 actcttgt GTCCC

[0525] CD63

[0526] agat CAGAA

[0527] CAG JI

[0528]

[0529] V > - - CCTGT

[0530] taatttct GAGGT actGtC GGCCG None None None None None gtagat CCGGC ATC CCTGT

[0531] taatttct GAGGT actGtC GGCCG None None None None None gtagat CCGGC ATC TCCCA

[0532] taatttct ATCTG actGtC TGTAG None None None None None gtagat TTAGC AGC ATACC

[0533] taatttct AAACT actGtC GTCCC None None None None None gtagat CAGAA CAG TCCCA

[0534] taatttct ATCTG aatttcta actGtC TGTAG ctctAgt None None None None gtagat TTAGC agai AGC TCCCA

[0535] taatttct ATCTG actGtC TGTAG None None None None None gtagat TTAGC AGC TCCCA

[0536] taatttct ATCTG actGtC TGTAG None None None None None gtagat TTAGC AGC

[0537] >

[0538]

[0539]

[0540] / . gtaatttctactcGCTTTAAGGCCGGTCCTAGCAAt tgtagatCAAGGACACCTACAACAAGCTGAtaatt

[0541] None tctactGGCTCACCTATTAGCGGCTAAGGtCgta gatCCTGTGAGGTGGCCGCCGGCATCtttttt

[0542] gtaatttetactcGCTCACCTATTAGCGGCTAAGGt tgtagatCAAGGACACCTACAACAAGCTGAtaatt

[0543] None tctactGGCTTTAAGGCCGGTCCTAGCAAtCgta gatCCTGTGAGGTGGCCGCCGGCATCtttttt

[0544] gtaatttotactcttgtagatATACCAAACT GT CCCCAG AACAGtaattctactGtCgtagatTCCCAATCTGTGT None AGTTAGCAGCtttttt

[0545] gtaatttctactcttgtagatTCCCAATCTGTGTAGTTA

[0546] iP> * GCAGCtaatttctactGtCgtagatATACCAAACTGT None CCCCAGAACAGtttttt

[0547] gtaattctactcttgtagatATACCAAACT GT CCCCAG AACAGtaattctactGtCgtagatTCCCAATCTGTGT None AGTTAGCAGCAaatttctactctAgtagattttttt

[0548] gtaattctactcttgtagatATACCAAACT GT CCCCAG AACAGGCTTTAAGGCCGGTCCTAGCAAtaattt

[0549] None ctactGtCgtagatTCCCAATCTGTGTAGTTAGCA GCGCTCACCTATTAGCGGCTAAGGtttttt

[0550] gtaatttctactcttgtagatATACCAAACTGTCCCCAG AACAGGCTCACCTATTAGCGGCTAAGGtaattt

[0551] None ctactGtCgtagatTCCCAATCTGTGTAGTTAGCA

[0552] GCGCTTTAAGGCCGGTCCTAGCAAtttttt

[0553]

[0554] V ATACC

[0555] taatttct AAACT

[0556] CD47,

[0557] Guide_#039 CS1. CS2 hg38 actcttgt GTCCC

[0558] CD63 agat CAGAA

[0559] CAG ATACC

[0560] taatttct AAACT

[0561] CD47,

[0562] Guide_#040 CS2. CS1 hg38 actcttgt GTCCC

[0563] CD63 agat CAGAA

[0564] CAG ATACC

[0565] taatttct AAACT

[0566] CD47,

[0567] Guide_#041 CS1, CS2 hg38 actcttgt GTCCC

[0568] CD63 agat CAGAA

[0569] CAG ATACC

[0570] taatttct AAACT

[0571] CD47,

[0572] Guide. #042 CS2. CS1 hg38 actcttgt GTCCC

[0573] CD63 agat CAGAA CAG

[0574]

[0575] CCTGT

[0576] taatttct GAGGT

[0577] CD81,

[0578] Guide_#043 CS1 hg38 actcttgt GGCCG

[0579] CD9 agat CCGGC

[0580] ATC TCCCA

[0581] taatttct ATCTG

[0582] CD63,

[0583] Guide_#044 CS2 hg38 actcttgt TGTAG

[0584] CD47 agat TTAGC

[0585] AGC CCTGT

[0586] taatttct GAGGT

[0587] CD81,

[0588] Guide_#045 CS1 hg38 actcttgt GGCCG

[0589] CD47 agat CCGGC

[0590] ATC TCCCA

[0591] taatttct ATCTG Guide_#046 CD63,

[0592] CS2 hg38 actcttgt TGTAG

[0593] CD9 agat TTAGC

[0594] AGC

[0595]

[0596] A TCCCA

[0597] taatttct ATCTG actGtC TGTAG None None None None None gtagat TTAGC AGO TCCCA

[0598] taatttct ATCTG actGtC TGTAG None None None None None gtagat TTAGC AGC TCCCA

[0599] taatttct ATCTG actGtC TGTAG None None None None None gtagat TTAGC AGC TCCCA

[0600] taatttct ATCTG actGtC TGTAG None None None None None gtagat TTAGC AGC CAAGG

[0601] taatttct ACACC actGtC TACAA None None None None None gtagat CAAGC TGA ATACC

[0602] taatttct AAACT actGtC GTCCC None None None None None gtagat CAGAA CAG ATACC

[0603] taatttct AAACT actGtC GTCCC None None None None None gtagat CAGAA CAG CAAGG

[0604] taatttct ACACC actGtC TACAA None None None None None gtagat CAAGC TGA

[0605] V

[0606]

[0607] A gtaatttctactcttgtagatGCTTTAAGGCCGGTCCT AGCAAATACCAAACTGTCCCCAGAACAGtaa None tttctactGtCgtagatGCTCACCTATTAGCGGCTA AGGTCCCAATCTGTGTAGTTAGCAGCtttttt

[0608] gtaatttctactcttgtagatGCTCACCTATTAGCGGC TAAGGATACCAAACTGTCCCCAGAACAGtaa None tttctactGtCgtagatGCTTTAAGGCCGGTCCTAG CAATCCCAATCTGTGTAGTTAGCAGCtttttt

[0609] gtaatttctactcGCTTTAAGGCCGGTCCTAGCAAt tgtagatATACCAAACTGTCCCCAGAACAGtaatt None tctactGGCTCACCTATTAGCGGCTAAGGtCgta gatTCCCAATCTGTGTAGTTAGCAGCtttttt

[0610] gtaatttctactcGCTCACCTATTAGCGGCTAAGGt tgtagatATACCAAACTGTCCCCAGAACAGtaatt None tctactGGCTTTAAGGCCGGTCCTAGCAAtCgta gatTCCCAATCTGTGTAGTTAGCAGCtttttt

[0611] gtaatttctactcttgtagatCCTGTGAGGTGGCCGCC GGCATCtaatttctactGtCgtagatCAAGGACACCT None ACAACAAGCTGAGCTTTAAGGCCGGTCCTA GCAAtttttt

[0612] gtaatttctactcttgtagatTCCCAATCTGTGTAGTTA GCAGCtaatttctactGtCgtagatATACCAAACTGT None CCCCAGAACAGGCTCACCTATTAGCGGCTA AGGttttt

[0613] gtaatttctactcttgtagatCCTGTGAGGTGGCCGCC GGCATCtaatttctactGtCgtagatATACCAAACTG

[0614] None TCCCCAGAACAGGCTTTAAGGCCGGTCCTA GCAAttttt

[0615] g t aattt ctactcttg tag atT CCCAAT CT GT GTAGTTA GCAGCtaatttctactGtCgtagatCAAGGACACCTA None CAACAAGCTGAGCTCACCTATTAGCGGCTA AGGtttttt

[0616]

[0617] > CAAGG

[0618] CD9, taatttct ACACC

[0619] CD81,

[0620] Guide_#047 CS1 hg38 actcttgt TACAA

[0621] CD47, agat CAAGG

[0622] CD63

[0623] TGA CAAGG CD9, taatttct ACACC

[0624] CD47,

[0625] Guide_#048 CS2 hg38 actcttgt TACAA

[0626] CD81, agat CAAGC

[0627] CD63

[0628] TGA ATACC CD47, taatttct AAACT

[0629] CD63,

[0630] Guide_#049 CS1 hg38 actcttgt GTCCC

[0631] CD9, agat CAGAA

[0632] CD81

[0633] CAG ATACC CD47, taatttct AAACT CD9,

[0634]

[0635] Guide_#050 CS2 hg38 actcttgt GTCCC CD63, agat CAGAA

[0636] CD81

[0637] CAG CAAGG

[0638] taatttct ACACC

[0639] CD9,

[0640] Guide_#101 CS1 hg38 actcttgt TACAA

[0641] CD81 agat CAAGC

[0642] TGA CAAGG

[0643] taatttct ACACC

[0644] CD9,

[0645] Guide_#102 CS2 hg38 actcttgt TACAA

[0646] CD81 agat CAAGC

[0647] TGA ATACC CD47, taatttct AAACT

[0648] CD63,

[0649] Guide_#103 CS1 hg38 actcttgt GTCCC

[0650] CD9, agat CAGAA

[0651] CD81

[0652] CAG

[0653]

[0654] A CCTGT ATACC TCCCA taatttct GAGGT aatttcta AAACT Aaatttc ATCTG actGtC GGCCG ctctAgt GTCCC tCctctC TGTAG None gtagat CCGGC agat CAGAA gGagat TTAGC ATC CAG AGC ATACC CCTGT TCCCA taatttct AAACT aatttcta GAGGT Aaatttc ATCTG actGtC GTCCC ctctAgt GGCCG tCctctC TGTAG None gtagat CAGAA agat CCGGC gGagat TTAGC CAG ATC AGC TCCCA CAAGG CCTGT taatttct ATCTG aatttcta ACACC Aaatttc GAGGT actGtC TGTAG ctctAgt TACAA tCctctC GGCCG None gtagat TTAGC agat CAAGC gGagat CCGGC AGC TGA ATC CAAGG TCCCA CCTGT taatttct ACACC aatttcta ATCTG Aaatttc GAGGT actGtC TACAA ctctAgt TGTAG tCctctC GGCCG None gtagat CAAGC agat TTAGC gGagat CCGGC TGA AGC ATC ACCAC

[0655] taatttct CTCAG actGtC TGCTC None None None None None gtagat AAGAA CAA ACCAC

[0656] taatttct CTCAG actGtC TGCTC None None None None None gtagat AAGAA CAA TCCCA CAAGG ACCAC taatttct ATCTG aatttcta ACACC Aaatttc CTCAG actGtC TGTAG ctctAgt TACAA tCctctC TGCTC None gtagat TTAGC agat CAAGC gGagat AAGAA AGC TGA CAA

[0657] V

[0658]

[0659] - - gtaatttctactcttgtagatCAAGGACACCTACAACA AGCTGAtaatttctactGtCgtagatCCTGTGAGGTG GCCGCCGGCATCaatttctactctAgtagatATACCA None AACTGTCCCCAGAACAGaaatttctCctctCgGag atTCCCAATCTGTGTAGTTAGCAGCGCTTTA AGGCCGGTCCTAGCAAtttttt gtaatttctactcttgtagatCAAGGACACCTACAACA AGCTGAtaatttctactGtCgtagatATACCAAACTG

[0660] T CCCCAGAACAGaatttctactctAgtagatCCTGTG None AGGTGGCCGCCGGCATCaaatttctCctctCgGag atTCCCAATCTGTGTAGTTAGCAGCGCTCAC CTATTAGCGGCTAAGGtttttt gtaatttctactcttgtagatATACCAAACT GT CCCCAG AACAGtaatttctactGtCgtagatTCCCAATCTGTGT AGTTAGCAGCaattctactctAgtagatCAAGGACA None CCTACAACAAGCTGAaaatttctCctctCgGagatC CTGTGAGGTGGCCGCCGGCATCGCTTTAA GGCCGGTCCTAGCAAtttttt gtaatttetactcttgtagatATACCAAACTGTCCCCAG jT> < AACAGtaattctactGtCgtagatCAAGGACACCTA CAACAAGCT GAaatttctactctAgtagatT CCCAAT None CTGTGTAGTTAGCAGCaaatttctCctctCgGagat CCTGTGAGGTGGCCGCCGGCATCGCTCAC CTATTAGCGGCTAAGGttttt gtaatttctactcttgtagatCAAGGACACCTACAACA AGCTGAtaatttctactGtCgtagatACCACCTCAGT None GCTCAAGAACAAGCTTTAAGGCCGGTCCTA GCAAtttttt

[0661] gtaatttctactcttgtagatCAAGGACACCTACAACA AGCTGAtaatttctactGtCgtagatACCACCTCAGT None GCTCAAGAACAAGCTCACCTATTAGCGGCT AAGGttttt gtaatttctactcttgtagatATACCAAACTGTCCCCAG AACAGtaatttctactGtCgtagatTCCCAATCTGTGT AGTTAGCAGCaatttctactctAgtagatCAAGGACA None CCTACAACAAGCTGAaaatttctCctctCgGagatA CCACCTCAGTGCTCAAGAACAAGCTTTAAG GCCGGTCCTAGCAAtttttt

[0662]

[0663] - 9 - ATACC

[0664] CD47, taatttct AAACT

[0665] CD63,

[0666] Guide_#104 CS2 hg38 actcttgt GTCCC

[0667] CD9, agat CAGAA

[0668] CD81

[0669] CAG CAAGG

[0670] taatttct ACACC

[0671] CD9,

[0672] Guide__#105 CS1 hg38 actcttgt TACAA

[0673] CD81 agat CAAGG

[0674] TGA CAAGG

[0675] taatttct ACACC

[0676] CD9,

[0677] Guide. #106 CS2 hg38 actcttgt TACAA

[0678] CD81 agat CAAGG

[0679] TGA ATACC CD47,

[0680]

[0681] taatttct AAACT > Fu CD63,

[0682] Guide. #107 CS1 hg38 actcttgt GTCCC

[0683] CD9, agat CAGAA

[0684] CD81

[0685] CAG ATACC CD47, taatttct AAACT

[0686] CD63,

[0687] Guide_#108 CS2 hg38 actcttgt GTCCC

[0688] CD9, agat CAGAA

[0689] CD81

[0690] CAG CTGAA

[0691] Guide. #109 NTC1, None hg38 taatttct GGTGT

[0692] NTC2, actcttgt CTGGC

[0693] NTC3, agat AGAGC

[0694] NTC4

[0695] TTA

[0696]

[0697] * TCCCA CAAGG ACCAC taatttct ATCTG aatttcta ACACC Aaatttc CTCAG actGtC TGTAG ctctAgt TACAA tCctctC TGCTC None gtagat TTAGC agat CAAGC gGagat AAGAA AGC TGA CAA ACCAC

[0698] taatttct CTCAG actGtC TGCTC None None None None None gtagat AAGAA CAA ACCAC

[0699] taatttct CTCAG actGtC TGCTC None None None None None gtagat AAGAA CAA TCCCA CAAGG ACCAC taatttct ATCTG aatttcta ACACC Aaatttc CTCAG actGtC TGTAG ctctAgt TACAA tCctctC TGCTC None gtagat TTAGC agat CAAGC gGagat AAGAA AGC TGA CAA TCCCA CAAGG ACCAC taatttct ATCTG aatttcta ACACC Aaatttc CTCAG actGtC TGTAG ctctAgt TACAA tCctctC TGCTC None gtagat TTAGC agat CAAGC gGagat AAGAA AGC TGA CAA CCCCA ACCGT ACGGC taatttct TCACA Aaatttc AGGCG Aaatttc GGTTC taatttct actGtC CTGAC tactctA ACGCT tCctctC GGTAA actTtC gtagat TGGAA gtagat AGCGT gGagat ACCTC gtagat ATC ACA GAG

[0700]

[0701] / =

[0702] gtaatttctactcttgtagatATACCAAACTGTCCCCAG AACAGtaatttctactGtCgtagatTCCCAATCTGTGT AGTTAGCAGCaatttctactctAgtagatCAAGGACA

[0703] None CCTACAACAAGCTGAaaatttctCctctCgGagatA CCACCTCAGTGCTCAAGAACAAGCTCACCT ATTAGCGGCTAAGGtttttt gtaatttctactcttgtagatCAAGGACACCTACAACA AGCTGAtaatttctactGtCgtagatACCACCTCAGT

[0704] None GCTCAAGAACAAGCTTTAAGGCCGGTCCTA GCAAtttttt gtaatttctactcttgtagatCAAGGACACCTACAACA

[0705] AGCT GAtaatttctactGtCgtagatACCACCT CAGT

[0706] None GCTCAAGAACAAGCTCACCTATTAGCGGCT AAGGtttttt gtaatttctactcttgtagatATACCAAACTGTCCCCAG w^> < AACAGtaatttctactGtCgtagatTCCCAATCTGTGT AGTTAGCAGCaatttctactGtAgtagatCAAGGACA

[0707] None CCTACAACAAGCTGAaaatttctCctctCgGagatA CCACCTCAGTGCTCAAGAACAAGCTTTAAG GCCGGTCCTAGCAAtttttt gtaatttctactcttgtagatATACCAAACTGTCCCCAG AACAGtaattctactGtCgtagatTCCCAATCTGTGT AGTTAGCAGCaatttctactctAgtagatCAAGGACA

[0708] None CCTACAACAAGCTGAaaatttctCctctCgGagatA CCACCTCAGTGCTCAAGAACAAGCTCACCT ATTAGCGGCTAAGGtttttt gTCCCAGAGCCACCGTTACACtaatttctactcttgt agatCTGAAGGTGTCTGGCAGAGCTTAtaatttct actGtCgtagatCCCCATCACACTGACTGGAAAT GCCTTC CAaatttctactctAgtagatACCGTAGGCGACGCTA CGGGAC GCGTACAAaatttctCctctCgGagatACGGCGGTT AGCAGC CGGTAAACCTCGACtaatttctactTtCgtagatACG C CTAAACGCCTGCAACCAGGCCTTCCGGGAC AGCAGCCtttttt

[0709]

[0710] CCTGT

[0711] CD81, taatttct GAGGT

[0712] CD9,

[0713] Guide_#110 None hg38 actcttgt GGCCG

[0714] CD47, agat CCGGC CD63

[0715] ATC

[0716] GATTG NTC5, taatttct GGCGC NTC6,

[0717] Guide_#111 CS1 hg38 actcttgt GTCGA

[0718] NTC7, agat CTACG

[0719] NTC8

[0720] GTG

[0721] CAAGG CD9, taatttct ACACC

[0722] CD81, Guide_#112 CS1 hg38 actcttgt TACAA CD63,

[0723]

[0724] agat CAAGG

[0725] CD47

[0726] TGA

[0727] GTCGG NTC9, taatttct TAGCG

[0728] NTC10,

[0729] Guide. #113 CS2 hg38 actcttgt AGCTA

[0730] NTC11, agat GCGAA

[0731] NTC12

[0732] ACC

[0733] ATACC CD47,

[0734] taatttct AAACT

[0735] CD63,

[0736] Guide_#114 CS2 hg38 actcttgt GTCCC

[0737] CD81, agat CAGAA

[0738] CD9

[0739] CAG

[0740]

[0741] A _ >

[0742] CAAGG ATACC TCCCA taatttct ACACC Aaatttc AAACT Aaatttc ATCTG taatttct actGtC TACAA tactctA GTCCC tCctctC TGTAG actTtC gtagat CAAGC gtagat CAGAA gGagat TTAGC gtagat TGA CAG AGC

[0743] GCTGA GGCGC GGTCG taatttct TCGAC Aaatttc GCTAT Aaatttc CGACC taatttct actGtC GCGGT tactctA ACTCG tCctctC TCTAC actTtC gtagat AAGCG gtagat CGAGC gGagat AATTC gtagat AAC CGT GCG

[0744] CCTGT TCCCA ATACC taatttct GAGGT Aaatttc ATCTG Aaatttc AAACT taatttct actGtC GGCCG tactctA TGTAG tCctctC GTCCC actTtC gtagat CCGGC gtagat TTAGC gGagat CAGAA gtagat ATC AGC CAG

[0745] TACGG AACGT AAGTC taatttct CGTCC Aaatttc GCCGT Aaatttc CATAC taatttct actGtC GACTT tactctA CTACG tCctctC CGTTC actTtC gtagat CGAGT gtagat GTAGG gGagat GTCGC gtagat GGA GCC GCG

[0746] TCCCA CCTGT CAAGG taatttct ATCTG Aaatttc GAGGT Aaatttc ACACC taatttct actGtC TGTAG tactctA GGCCG tCctctC TACAA actTtC gtagat TTAGC gtagat CCGGC gGagat CAAGC gtagat AGC ATC TGA

[0747]

[0748] / — -kgTCCCAGAGCCACCGTTACACtaatttetactcttgt agatCCTGTGAGGTGGCCGCCGGCATCtaatttctactGtCgtagatCAAGGACACCTACAACAAGCTCCAGG TGAAaattctactctAgtagatATACCAAACTGTCC CTGCTG CCAGAACAGAaattctCctctCgGagatTCCCAAT TCCCGG CTGTGTAGTTAGCAGCtaatttctactTtCgtagatAC A GCTAAACGCCTGCAACCAGTCCAGGCTGCT GTCCCGGAtttttt gTCCCAGAGCCACCGTTACACtaatttctactcttgt agatGATTGGGCGCGTCGACTACGGTGtaatttc tactGtCgtagatGCTGATCGACGCGGTAAGCG GCTCTC AACAaatttctactctAgtagatGGCGCGCTATACTC ATAGAT GCGAGCCGTAaatttctCctctCgGagatGGTCGC GTCCTC GACCTCTACAATTCGCGtaatttctactTtCgtagatA C CGCTAAACGCCTGCAACCAGGCTCTCATAG ATGTCCTCCGCTTTAAGGCCGGTCCTAGCA Atttttt gTCCCAGAGCCACCGTTACACtaatttctactcttgt agatCAAGGACACCTACAACAAGCTGAtaatttct actGtCgtagatCCTGTGAGGTGGCCGCCGGCA CTGGTA T CAaatttctactctAgtagatT CCCAAT CT GT GT AGT CTCTTC TAGCAGCAaatttctCctctCgGagatATACCAAAC CGTGAA TGTCCCCAGAACAGtaatttctactTtCgtagatACG G CTAAACGCCTGCAACCAGCTGGTACTCTTC CGTGAAGGCTTTAAGGCCGGTCCTAGCAAtt tttt gTCCCAGAGCCACCGTTACACtaatttetactcttgt agatGTCGGTAGCGAGCTAGCGAAACCtaatttc tactGtCgtagatTACGGCGTCCGACTTCGAGTG GAAGA GAAaatttctactctAgtagatAACGTGCCGTCTACG GTACCA GTAGGGCCAaatttctCctctCgGagatAAGTCCAT GCTCTT ACCGTTCGTCGCGCGtaatttctactTtCgtagatAC CG GCTAAACGCCTGCAACCAGGAAGAGTACCA GCTCTTCGGCTCACCTATTAGCGGCTAAGG tttttt gTCCCAGAGCCACCGTTACACtaatttctactcttgt agatATACCAAACTGTCCCCAGAACAGtaatltcl actGtCgtagatTCCCAATCTGTGTAGTTAGCAG TACCAG CAaatttctactctAgtagatCCTGTGAGGTGGCCGC CTCTTC CGGCATCAaatttctCctctCgGagatCAAGGACAC GAGGA CTACAACAAGCTGAtaatttctactTtCgtagatACGC AT TAAACGCCTGCAACCAGTACCAGCTCTTCG

[0749]

[0750] AGGAATGCTCACCTATTAGCGGCTAAGGtttttt pre-crRNA for cell surface receptors

[0751] pre- crRNA Full vector sequence

[0752] ID

[0753] ggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagt gaacggatctcgacggtatcggttaacttttaaaagaaaaggggggattggggggtcagtgcaggggaaagaa tagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttatcgat gagtaattcatacaaaaggactcgcccctgccttggggaatcccagggaccgtcgttaaactcccactaacgtag aacccagagatcgctgcgttcccgccccctcacccgccgctctcgtcatcactgaggtggagaagagcatgcgt gaggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtc ggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcc tttttcccgagggtgggggagaaccgatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgcc gccagaacacaggtaagtgccgtgtgtggttcccgcgggcctggcctcttacgggttatggccctgcgtgcct tgaattacttccacgcccctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagt tcgaggcttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcttgggcgctggggccgccgc gtgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctg ctgcgacgctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtattcggtttttggggc cgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggcc accgagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatc gccccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagtggccgcttcccggc cctgctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaa ggaaaagggcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctc gattagttctcaagcttttggagtacgtcgtctttaggttgggggaggggttttatgcgatggagtttccccacactg agtgggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggatc ttggttcattctcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtcgtgaccctagcgctacctct Guide agagccaccatgaccggtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggccgtacgc #025 ~ accctcgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatccggaccgccacatcgagcgg gtcaccgagctgcaagaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacgac ggcgccgccgtggcgtctggaccacgccggagagcgtcgaagcgggggcggtgttcgccgagatcggccc gcgcatggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcctcctggcgccgcacc ggcccaaggagcccgcgtggttcctggccaccgtcggagtctcgcccgaccaccagggcaagggtctgggc aggccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcg ccccgcaacctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgc gcacctggtgcatgacccgcaagcccggtgcctgaacgcgttaagtcgacaatcaactctggattacaaaatttg tgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgct attgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttg tcaggcaacgtggcgtggtgtgcactgtgttgctgacgcaacccccactggttggggcattgccaccacctgtca gctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgct ggacaggggctcggctgttgggcactgacaatccgtggtgtgtcggggaaatcatcgtcctttccttggctgct cgcctgtgttgccacctggattcgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttc cttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccct ttgggccgcctccccgcgtcgactttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaaga aaagggggactggaagggctaattcactcccaacgaagacaagatttcaccattatcgtttcagacccacctccc aaccccgaggggacccagagagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttaga gagataatagaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtataatttcttg ggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggc tttatatatcttgtggaaaggacgaggtaccgtaatttctactcttgtagatCTGAAGGTGTCTGCiCAGAGCTTAtaatttctactGtCgtagatCCCCATCACACTGACTGGAAATCttttttgaat

[0754]

[0755] tcaacttggcgtaactagatcttgagacactgctttttgcttgtactgggtctctctggttagaccagatctgagcctg ggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtg cccgtctgttgtgtgactctggtaactagagatccctcaacccttttagtcagtgtggaaaatctctagcagtacgta tagtagttcatgtcatctattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttat tgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagtt gtggtttgtccaaactcatcatgtatcttatcatgtctggctctagctatcccgcccctaactccgcccatcccgccc ctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcc tcggcctctgagctatccagaagtagtgaggaggcttttttggaggcctagggacgtacccaattcgccctatag tgagtcgtttacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaactta atcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaa cagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggtta gcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgt tcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgac cccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagaggtttttcgccctttgacgttg gagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgattt ataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaa atattaacgcttacaatttaggtggcacttttcgggaaatgtgcgcggaacccctatttgtttatttttctaaatacattc aaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattc aacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaag taaaagatgctgaagatcagttggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttga gagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtatt gacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgactggttgagtactcacc.agtc.aca gaaaagcatttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcgg ccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaa Guide ctcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctga #025 ’ gcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagact ggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttatgctgataaatc tggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtagccctcccgtatcgtagt tatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactga ttaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggat ctaggtgaagatcctttttgataatctcatgaccaaatcccttaacgtgagttttcgttccactgagcgtcagacccc gtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccg ctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgc agataccaaatactgttcttctagtgtgccgtagtaggccaccacttcaagaactctgtagcaccgcctacatacc tcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacg atagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaac gacctacaccgaactggatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcg gacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcct ggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggg cctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcct gcgttatcccctgattctgtggataaccgtattaccgccttgagtgagctgataccgctcgccgcagccgaacga ccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgccctccccgcgcgt tggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaa tgtgagttagctcactcataggcaccccaggcttacactttatgcttccggctcgtatgttgtgtggaattgtgagc ggataacaatttcacacaggaaacagctatgaccatgatacgccaagcgcgcaattaaccctcactaaagggaa caaaagctggagctgcaagcttaatgtagtcttatgcaatactcttgtagtctgcaacatggtaacgatgagttag caacatgccttacaaggagagaaaaagcaccgtgcatgccgattggtggaagtaaggtggtacgatcgtgcctt attaggaaggcaacagacgggtctgacatgattggacgaaccactgaatgccgcattgcagagatattgtattta agtgcctagctcgatacataaacgggtctctctggttagaccagatctgagcctgggagctctctggctaactagg

[0756]

[0757] gaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctgg taactagagatcctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaag cgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcga ggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgaga gcgtcgtataagcgggggagaatagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaa atataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaaca tcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggatcagaaaacttagatcattat ataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaaga tagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggaggagg Guide agatatgagggacaattggagaagtgaattatataaatataaagtagtaaaattgaaccattaggagtagcaccca #025 ' ccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttctt gggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggt atagtgcagcagcagaacaatttgctgagggtattgaggcgcaacagcatctgttgcaactcacagtctggggc atcaagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttgggg ttgctctggaaaactcattgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaacagatttg gaatcacacgaccggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaa tcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggttta acataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagttttgct gtacttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgagg

[0758]

[0759] ggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagt gaacggatctcgacggtatcggttaacttttaaaagaaaaggggggattggggggtcagtgcaggggaaagaa tagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttatcgat gagtaattcatacaaaaggactcgcccctgccttggggaatcccagggaccgtcgttaaactcccactaacgtag aacccagagatcgctgcgttcccgccccctcacccgccgctctcgtcatcactgaggtggagaagagcatgcgt gaggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtc ggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcc tttttcccgagggtgggggagaaccgatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgcc gccagaacacaggtaagtgccgtgtgtggtcccgcgggcctggcctctttacgggttatggcccttgcgtgcct tgaattacttccacgcccctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagt tcgaggcttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcttgggcgctggggccgccgc gtgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctg ctgcgacgctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtattcggttttggggc cgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggcc accgagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatc gccccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagtggccgcttcccggc cctgctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaa ggaaaagggcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctc gattagttctcaagctttggagtacgtcgtctttaggttgggggaggggttttatgcgatggagtttccccacactg agtgggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggatc ttggttcattctcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtcgtgaccctagcgctacctct agagccaccatgaccggtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggccgtacgc accctcgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatccggaccgccacatcgagcg ggtcaccgagctgcaagaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacga Guide cggcgccgccgtggcgtctggaccacgccggagagcgtcgaagcgggggcggtgttcgccgagatcggcc #026 ’ cgcgcatggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcctcctggcgccgcac cggcccaaggagcccgcgtggttcctggccaccgtcggagtctcgcccgaccaccagggcaagggtctggg caggccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgcctcctggagacctccgc gccccgcaacctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggacc gcgcacctggtgcatgacccgcaagcccggtgcctgaacgcgttaagtcgacaatcaactctggattacaaaat ttgtgaaagattgactggtattctaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcat gctattgcttcccgtatggctttcattttctcctcctgtataaatcctggttgctgtctctttatgaggagttgtggcccg ttgtcaggcaacgtggcgtggtgtgcactgtgttgctgacgcaacccccactggttggggcattgccaccacctg tcagctcctttccgggactttcgctttccccctccctatgccacggcggaactcatcgccgcctgccttgcccgct gctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggct gctcgcctgtgttgccacctggatcgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggac cttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatct ccctttgggccgcctccccgcgtcgactttaagaccaatgacttacaaggcagctgtagatcttagccactttttaa aagaaaagggggactggaagggctaattcactcccaacgaagacaagatttcaccattatcgtttcagacccac ctcccaaccccgaggggacccagagagggcctatttcccatgattccttcatatttgcatatacgatacaaggctg ttagagagataattagaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtataatt tcttgggtagtttgcagtttaaaattatgtttaaaatggactatcatatgcttaccgtaacttgaaagtattcgatttct tggctttatatatcttgtggaaaggacgaggtaccgtaatttctactcttgtagatCAAGGACACCTACA ACAAGCTGAtaatttctactGtCgtagatCCTGTGAG<jTG<:iCCGCCG(:rCATCtttttt gaattcaacttggcgtaactagatcttgagacactgctttttgcttgtactgggtctctctggttagaccagatctgag cctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagt gtgtgcccgtctgttgtgtgactctggtaactagagatccctcgacccttttagtcagtgtggaaaatctctagcagt acgtatagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaact tgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcatt

[0760]

[0761] ctagttgtggtttgtccaaactcacaatgtatcttatcatgtctggctctagctatcccgcccctaactccgcccatcc cgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgag gccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctagggacgtacccaattcgc cctatagtgagtctattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacc caacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgccc ttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtg gtggtacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctc gccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgattagtgctttacggca cctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgataacggtttttcgcccttt gacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattc ttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattt taacaaaatattaacgcttacaatttaggtggcactttcgggaaatgtgcgcggaacccctatttgtttatttttctaa atacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtat gagtattcaacatttccgtgtcgcccttattcccttttgcggcattttgccttcctgtttttgctcacccagaaacgct ggtgaaagtaaaagatgctgaagatcagtgggtgcacgagtgggttacatcgaactggatctcaacagcggtaa gatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtatt atcccgtattgacgccgggcaagagcaactcggtcgccgcatac.actattctc.agaatgacttggttgagtactc accagtcacagaaaagcacttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtga taacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatggg ggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacacc acgatgccttagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaaca attaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttatt gctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtagccctcc Guide cgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagatagg #026 ’ tgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaa tttaaaaggatctaggtgaagatcctttgataatctcatgacaaaatccctaacgtgagtttcgttccactgagc gtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgctgcaaacaaa aaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttc agcagagcgcagataccaaatactgttcttctagttagccgtagttaggccaccacttcaagaactctgtagcacc gcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttgg actcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagct tggagcgaacgacctacaccgaacgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagg gagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagg gggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgattttgtgatgctcgtca ggggggcgagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctca catgttctttcctgcgtatcccctgattctgtggataaccgtattaccgccttgagtgagctgataccgctcgccgc agccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaacccctct ccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgc aacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtg gaattgtgagcggataacaatttcacacaggaaacagctatgaccatgttacgccaagcgcgcaattaaccctca ctaaagggaacaaaagctggagctgcaagcttaatgtagtcttatgcaatactcttgtagtcttgcaacatggtaac gatgagttagcaacatgccttacaaggagagaaaaagcaccgtgcatgccgattggtggaagtaaggtggtac gatcgtgccttattaggaaggcaacagacgggtctgactggattggacgaaccactgaattgccgcattgcaga

[0762]

[0763] gatattgtatttaagtgcctagctcgatacataaacgggtctctctggttagaccagatctgagcctgggagctctct ggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgtt gtgtgactctggtaactagaatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacag ggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacgg caagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagat gggtgcgagagcgcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggg gaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcc tgttagaaac.atc.agaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggatcaaagaac ttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagc tttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagac Guide ctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagaaaaattgaaccattagg #026 agtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgtt ccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagaca attattgtctggtatagtgcagcagcagaacaatttgctgaggctattgaggcgcaacagcatctgtgcaactcac agtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctg gggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctg gaacagatttggaatcacacgactggatggagtgggacagagaaattaacaatacacaagcttaatacactcct taatgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgt ggaattggtttaacataacaaatggctgtggtatataaaattattcataatgatagtaggaggctggtaggtttaag aatagttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaa ccccgagg

[0764]

[0765] ggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagt gaacggatctcgacggtatcggttaacttttaaaagaaaaggggggattggggggtcagtgcaggggaaagaa tagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttatcgat gagtaattcatacaaaaggactcgcccctgccttggggaatcccagggaccgtcgttaaactcccactaacgtag aacccagagatcgctgcgttcccgccccctcacccgccgctctcgtcatcactgaggtggagaagagcatgcgt gaggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtc ggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcc tttttcccgagggtgggggagaaccgatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgcc gccagaacacaggtaagtgccgtgtgtggtcccgcgggcctggcctctttacgggttatggcccttgcgtgcct tgaattacttccacgcccctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagt tcgaggcttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcttgggcgctggggccgccgc gtgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctg ctgcgacgctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtattcggttttggggc cgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggcc accgagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatc gccccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagtggccgcttcccggc cctgctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaa ggaaaagggcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctc gattagttctcaagctttggagtacgtcgtctttaggttgggggaggggttttatgcgatggagtttccccacactg agtgggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggatc ttggttcattctcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtcgtgaccctagcgctacctct agagccaccatgaccggtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggccgtacgc accctcgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatccggaccgccacatcgagcg ggtcaccgagctgcaagaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacga Guide cggcgccgccgtggcgtctggaccacgccggagagcgtcgaagcgggggcggtgttcgccgagatcggcc #027 ’ cgcgcatggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcctcctggcgccgcac cggcccaaggagcccgcgtggttcctggccaccgtcggagtctcgcccgaccaccagggcaagggtctggg caggccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgcctcctggagacctccgc gccccgcaacctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggacc gcgcacctggtgcatgacccgcaagcccggtgcctgaacgcgttaagtcgacaatcaactctggattacaaaat ttgtgaaagattgactggtattctaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcat gctattgcttcccgtatggctttcattttctcctccttgtataaatcctggtgctgtctctttatgaggagttgtggccc gttgtcaggcaacgtggcgtggtgtgcactgtgttgctgacgcaacccccactggttggggcattgccaccacct gtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgc tgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttcctggc tgctcgcctgtgttgccacctggatcgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggac cttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatct ccctttgggccgcctccccgcgtcgactttaagaccaatgacttacaaggcagctgtagatcttagccactttttaa aagaaaagggggactggaagggctaattcactcccaacgaagacaagatttcaccattatcgtttcagacccac ctcccaaccccgaggggacccagagagggcctatttcccatgattccttcatatttgcatatacgatacaaggctg ttagagagataattagaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtataatt tcttgggtagtttgcagttttaaaattatgtttaaaatggactatcatatgcttaccgtaacttgaaagtattcgatttct tggctttatatatcttgtggaaaggacgaggtaccgtaatttctactcttgtagatcaaggacacctacaacaagctg ataatttctactgtcgtagatcctgtgaggtggccgccggcatcaaatttctactctatagatttttttgaattcaagct ggcgtaactagatcttgagacactgctttttgcttgtactgggtctctctggttagaccagatctgagcctgggagct ctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtc tgttgtgtgactctggtaactagagatccctcagacccttttatcagtgtggaaaatctctagcagtacgtatagtag ttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttatgcag cttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcattttttcactgcattctagttgtggtt

[0766]

[0767] tgtccaaactcatcaatgtatctatcatgtctggctctagctatcccgcccctaactccgcccatcccgcccctaact ccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctcggc ctctgagctattccagaagtagtgaggaggcttttttggaggcctagggacgtacccaattcgccctatagtgagt cgtattacgcgcctcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgc cttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagtt gcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcg cagcggaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgc cggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgacccca aaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcccctttgacgttggagt ccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataag ggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatatta acgcttacaatttaggtggcacttttcggggaaatgtgcgcgaacccctatttgtttatttttctaaatacattcaaatat gtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacattt ccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaag atgctgaagatcagttgggtgcacagtgggttacatcgaactggatctcaacagcggtaagatccttgagagtttt cgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgcc gggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaag catcttacggatgcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaactt acttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgcct tgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatg caacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatgga ggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggttattgctgataaatctggagcc ggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctccgtatcgtagttatctaca cgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcat Guide tggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtga #027 ’ agatcctttttgataatctcatgaccaaaatcccttacgtgagttttcgttccactgagcgtcagaccccgtagaaaa gatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagc ggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagatacca aatactgttcttctagtgtagccgtagtaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctg ctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttac cggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctac accgaactgagatacctaagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggt atccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatcttt atagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatga aaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcc cctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgc agcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgccgttggccgattc attaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttag ctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaa tttcacacaggaaacagctatgaccatgattacgccaaggcgcaattaaccctcactaaagggaacaaaagctg gagctgcaagcttaatgtagtcttatgcaatactcttgtagtcttgcaacatggtaacgatgagttagcaacatgcct tacaaggagagaaaaagcaccgtgcatgccgattggtggaagtaaggtggtacgatcgtgccttattaggaagg caacagacgggtctgacatggattggacaaccactgaatgccgcattgcagagatattgtattaagtgcctagc tcgatacataaacgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccact gcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagag atccctcagaccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaagcgaaaggg aaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggc gactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtat

[0768]

[0769] taaggggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaa aacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggct gtagacaaatactgggacagctacaaccatcccttcagacaggatcagaagaactagacattatataatacagta gcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaaga gcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggaggaggagatatgagg gacaattggagaagtgaattatataaatataaagtagtaaaaattgaacattaggagtagcacccaccaaggcaa agagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagcagc Guide aggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagc #027 agcagaacaatttgctgagggctattgagggcaacagcatctgttgcaactcacagtctggggcatcaagcagc tccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaa aactcattgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaacagatttggaatcacacg acctggatggaggggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaacc agcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaa ttggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtatttctata gtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgagg

[0770]

[0771] ggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagt gaacggatctcgacggtatcggttaacttttaaaagaaaaggggggattggggggtcagtgcaggggaaagaa tagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttatcgat gagtaattcatacaaaaggactcgcccctgccttggggaatcccagggaccgtcgttaaactcccactaacgtag aacccagagatcgctgcgttcccgccccctcacccgccgctctcgtcatcactgaggtggagaagagcatgcgt gaggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtc ggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcc tttttcccgagggtgggggagaaccgatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgcc gccagaacacaggtaagtgccgtgtgtggtcccgcgggcctggcctctttacgggtatggcccttgcgtgcct tgaattacttccacgcccctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagt tcgaggcttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcttgggcgctggggccgccgc gtgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctg ctgcgacgctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtattcggttttggggc cgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggcc accgagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatc gccccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagtggccgcttcccggc cctgctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaa ggaaaagggcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctc gattagttctcaagctttggagtacgtcgtctttaggttgggggaggggttttatgcgatggagtttccccacactg agtgggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggatc ttggttcattctcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtcgtgaccctagcgctacctct agagccaccatgaccggtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggccgtacgc accctcgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatccggaccgccacatcgagcgg gtcaccgagctgcaagaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacgacg Guide gcgccgccgtggcgtctggaccacgccggagagcgtcgaagcgggggcggtgttcgccgagatcggcccg #028 ’ cgcatggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcctcctggcgccgcaccgg cccaaggagcccgcgtggttcctggccaccgtcggagtctcgcccgaccaccagggcaagggtctgggcag gccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgcc ccgcaacctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcg cacctggtgcatgacccgcaagcccggtgcctgaacgcgttaagtcgacaatcaactctggattacaaaatttg tgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgct attgcttcccgtatggcttcatttctcctcctgtataaatcctggttgctgtctctttatgaggagttgtggcccgttg tcaggcaacgtggcgtggtgtgcactgtgttgctgacgcaacccccactggttggggcattgccaccacctgtca gctcctttccgggactttcgctttccccctccctatgccacggcggaactcatcgccgcctgccttgcccgctgct ggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgct cgcctgtgttgccacctggatcgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttc cttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccct ttgggccgcctccccgcgtcgactttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaaga aaagggggactggaagggctaattcactcccaacgaagacaagatttcaccattatcgtttcagacccacctccc aaccccgaggggacccagagagggcctattcccatgattccttcatatttgcatatacgatacaaggctgttaga gagataattagaattaattgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtataatttcttg ggtagtttgcagtttaaaattatgttaaaatggactatcatatgctaccgtaacttgaaagtattcgatttcttggc tttatatatcttgtggaaaggacgaggtaccgtaatttctactcttgtagatcaaggacacctacaacaagctgagct ttaaggccggtcctagcaataatttctactgtcgtagatcctgtgaggtggccgcggcatcgctcacctatagcg gctaaggttttttgaattcaagcttggcgtaactagatcttgagacactgctttttgcttgtactgggtctctctggttag accagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagt gcttcaagtagtgtgtgcccgtctgttgtgtgactctggaactagagatccctcagacccttttagtcagtgtggaa aatctctagcagtacgtatagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagaga gtgagaggaacttgttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcat

[0772]

[0773] ttttttcactgcattctagttgtgttgtccaaactcatcaatgtatcttatcatgtctggctctagctatcccgcccctaa ctccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttattatgc agaggccgaggccgcctcggcctctgagctatccagaagtagtgaggaggcttttttggaggcctagggacgt acccaattcccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaacc ctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgca ccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcataagcgc gcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctccttcgctttcttcc cttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctcccttagggttccgatttagtg ctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtggccatcgccctgatagacggtt tttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatct cggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatta acgcgaattttaacaaaatattaacgcttacaattaggtggcacttttcggggaaatgtgcgcggaacccctatttgt ttattttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaag gaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcaccca gaaacgctggtgaaagtaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaac agcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcactttaaagtctgctatgtggc gcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttg agtactcacagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccat gagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaa catgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagctga caccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactactactctagcttcccgg caacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctg gtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagc.ctggggccagatggtaag ccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctga Guide gataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttc #028 ’ atttttaatttaaaaggatctaggtgaagatcctttttataatctcatgaccaaaatcccttaacgtgagttttcgttccac tgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaa acaaaaaaaccaccgctaccagcggtggtttgttgccggatcaagagctaccaactcttttccgaaggtaactg gcttcagcagagcgcagataccaaaactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgta gcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccg ggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagc ccagcttggagcgaacacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttccc gaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagctt ccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgt cgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggcctt ttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgct cgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagccccaatacgcaaac cgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagt gagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtat gttgtgtggaatgtgagcggataacaatttcacacaggaaacgctatgaccatgattacgccaagcgcgcaatt aaccctcactaaagggaacaaaagctggagctgcaagcttaatgtagtcttatgcaatactcttgtagtcttgcaac atggtaacgatgagttagcaacatgccttacaaggagagaaaaagcaccgtgcatgccgattggtggaagtaag gtggtacgatcgtgccttattaggaaggcacagacgggtctgacatggattggacgaaccactgaattgccgcat tgcagagatatgtatttaagtgcctagctcgatacataaacgggtctctctggttagaccagatctgagcctggga gctctctggctaactagggaacccactgcttaagcctcaataaagcttgcctgagtgcttcaagtagtgtgtgccc gtctgtgtgtgatctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgccc gaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagcgc gcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggag

[0774]

[0775] agagatggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggcc agggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatc ctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatocctcagacaggatcag aagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaa ggaagctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatct tcagacctggaggaggagatatgagggacaattggagaagtgaattatataatataaagtagtaaaaattgaacc attaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggag Guide_ ctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggcc #028 agacaattattgtctggtatagtgcagcagcagacaatttgctgagggctattgaggcgcaacagcatctgttgca actcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacag ctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataa atctctggaacagattggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaata cactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaatagataaatgggca agtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtag tttaagaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacc tcccaaccccgagg

[0776]

[0777] ggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagt gaacggatctcgacggtatcggttaacttttaaaagaaaaggggggattggggggtcagtgcaggggaaagaa tagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttatcgat gagtaattcatacaaaaggactcgcccctgccttggggaatcccagggaccgtcgttaaactcccactaacgtag aacccagagatcgctgcgttcccgccccctcacccgccgctctcgtcatcactgaggtggagaagagcatgcgt gaggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtc ggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcc tttttcccgagggtgggggagaaccgatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgcc gccagaacacaggtaagtgccgtgtgtggtcccgcgggcctggcctctttacgggtatggcccttgcgtgcct tgaattacttccacgcccctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagt tcgaggctgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcttgggcgctggggccgccgc gtgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctg ctgcgacgctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtattcggttttggggc cgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggcc accgagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatc gccccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagtggccgcttcccggc cctgctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaa ggaaaagggcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctc gattagttctcaagctttggagtacgtcgtctttaggttgggggaggggttttatgcgatggagtttccccacactg agtgggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggatc ttggttcattctcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtcgtgaccctagcgctacctct agagccaccatgaccggtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggccgtacgc accctcgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatccggaccgccacatcgagcg ggtcaccgagctgcaagaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacgac Guide ggcgccgccgtggcgtctggaccacgccggagagcgtcgaagcgggggcggtgttcgccgagatcggccc #029 ’ gcgcatggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcctcctggcgccgcacc ggcccaaggagcccgcgtggttcctggccaccgtcggagtctcgcccgaccaccagggcaagggtctgggc aggccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcg ccccgcaacctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgc gcacctggtgcatgacccgcaagcccggtgcctgaacgcgttaagtcgacaatcaactctggattacaaaatttg tgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgct attgcttcccgtatggctttcattttctcctcctgtataaatcctggttgctgtctctttatgaggagttgtggcccgttg tcaggcaacgtggcgtggtgtgcactgtgttgctgacgcaacccccactggttggggcattgccaccacctgtca gctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgct ggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgct cgcctgtgttgccacctggatcgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggacctc cttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccct ttgggccgcctccccgcgtcgactttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaaga aaagggggactggaagggctaattcactcccaacgaagacaagatttcaccattatcgtttcagacccacctccc aaccccgaggggacccagagagggcctattcccatgattccttcatatttgcatatacgatacaaggctgttaga gagataattagaattaattgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtataatttcttg ggtagtttgcagtttaaaattatgttaaaatggactatcatatgcttaccgtaactgaaagtattcgatttcttggc tttatatatcttgtggaaaggacgaggtaccgtaatttctactcttgtagatcaaggacacctacaacaagctgagct cacctattagcggctaaggtaatttctactgtcgtagatcctgtgaggtggccgcggcatcgctttaaggccggtc ctagcaattttttgaattcaagcttggcgtaactagatcttgagacactgctttttgcttgtactgggtctctctggttag accagatctgagcctgggagctctctggctaactagggaacccactgctaagcctcaataaagctgccttgagt gcttcaagtagtgtgtgcccgtctgttgtgtgactctggaactagagatccctcagacccttttagtcagtgtggaaa atctctagcagtacgtatagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagag tgagaggaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcattt

[0778]

[0779] ttttcactgcattctagttgtgtttgtccaaactcatcaatgtatctatcatgtctggctctagctatcccgcccctaact ccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttattatgca gaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctagggacgta cccaattcccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccct ggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcac cgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcg cgggtgtggtggtacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttccc ttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgc tttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtggccatcgccctgatagacggttt ttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctc ggtctattctttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaa cgcgaattttaacaaaatattaacgcttacaattaggtggcacttttcggggaaatgtgcgcggaacccctatttgtt tatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaagg aagagtatgagtattcaacatttccgtgtcgcccttattcccttttgcggcattttgccttcctgtttttgctcacccag aaacgctggtgaaagtaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaaca gcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcg cggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttga gtactcacagtcacagaaaagcatctacggatggcatgacagtaagagaattatgcagtgctgccataaccatg agtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaac atgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagctgac accacgatgcctgtagcaatggcaacaacgttgcgcaaactataactggcgaactactactctagcttcccggc aacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggt ttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcatgcagcctggggccagatggtaagcc ctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagat Guide aggtgcctcactgattaagcattggtaactgtcagaccaagttactcatatatacttagattgatttaaaacttcattt #029 ’ ttaatttaaaaggatctaggtgaagatcctttttataatctcatgaccaaaatcccttaacgtgagttttcgttccactga gcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaaca aaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggct tcagcagagcgcagataccaaaactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagca ccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggtt ggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagccca gcttggagcgaacacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaa gggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagctccag ggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttgtgatgtcgtca ggggggcggagcctatggaaaaacgccagcaacgcggcctttacggttcctggccttttgctggccttttgctc acatgttctttcctgcgtatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccg cagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagccccaatacgcaaaccgcctc tccccgcgcgtggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcg caacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgt ggaatgtgagcggataacaatttcacacaggaaacgctatgaccatgatacgccaagcgcgcaattaaccctc actaaagggaacaaaagctggagctgcaagcttaatgtagtcttatgcaatactcttgtagtcttgcaacatggtaa cgatgagtagcaacatgcctacaaggagagaaaaagcaccgtgcatgccgatggtggaagtaaggtggtac gatcgtgccttattaggaaggcacagacgggtctgacatggattggacgaaccactgaattgccgcattgcaga gatattgtatttaagtgcctagctcgatacataaacgggtctctctggttagaccagatctgagcctgggagctctct ggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgtt gtgtgatctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacag ggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacgg caagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagat ggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggg

[0780]

[0781] gaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcc tgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatccctcagacaggatcagaagaac ttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagc tttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagac ctggaggaggagatatgagggacaattggagaagtgaattatataatataaagtagtaaaaattgaaccattagg agtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgtt ccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagaca Guide attattgtctggtatagtgcagcagcagacaatttgctgagggctattgaggcgcaacagcatctgttgcaactcac #029 agtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctg gggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctg gaacagattggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactcct taattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgt ggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtagtttaaga atagttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaac cccgagg

[0782]

[0783] ggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagt gaacggatctcgacggtatcggttaacttttaaaagaaaaggggggattggggggtcagtgcaggggaaagaa tagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttatcgat gagtaattcatacaaaaggactcgcccctgccttggggaatcccagggaccgtcgttaaactcccactaacgtag aacccagagatcgctgcgttcccgccccctcacccgccgctctcgtcatcactgaggtggagaagagcatgcgt gaggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtc ggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcc tttttcccgagggtgggggagaaccgatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgcc gccagaacacaggtaagtgccgtgtgtggtcccgcgggcctggcctctttacgggtatggcccttgcgtgcct tgaattacttccacgcccctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagt tcgaggcttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcttgggcgctggggccgccgc gtgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctg ctgcgacgctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtattcggttttggggc cgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggcc accgagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatc gccccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagtggccgcttcccggc cctgctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaa ggaaaagggcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctc gattagttctcaagctttggagtacgtcgtctttaggttgggggaggggttttatgcgatggagtttccccacactg agtgggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggatc ttggttcattctcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtcgtgaccctagcgctacctct agagccaccatgaccggtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggccgtacgc accctcgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatccggaccgccacatcgagcgg gtcaccgagctgcaagaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacgac Guide ggcgccgccgtggcgtctggaccacgccggagagcgtcgaagcgggggcggtgttcgccgagatcggccc #030 ’ gcgcatggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcctcctggcgccgcacc ggcccaaggagcccgcgtggttcctggccaccgtcggagtctcgcccgaccaccagggcaagggtctgggc aggccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcg ccccgcaacctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgc gcacctggtgcatgacccgcaagcccggtgcctgaacgcgttaagtcgacaatcaactctggattacaaaatttg tgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgct attgcttcccgtatggcttcatttctcctcctgtataaatcctggttgctgtctctttatgaggagttgtggcccgttg tcaggcaacgtggcgtggtgtgcactgtgttgctgacgcaacccccactggttggggcattgccaccacctgtca gctcctttccgggactttcgctttccccctccctatgccacggcggaactcatcgccgcctgccttgcccgctgct ggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgct cgcctgtgttgccacctggatcgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttc cttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccct ttgggccgcctccccgcgtcgactttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaaga aaagggggactggaagggctaattcactcccaacgaagacaagatttcaccattatcgtttcagacccacctccc aaccccgaggggacccagagagggcctattcccatgattccttcatatttgcatatacgatacaaggctgttaga gagataattagaattaattgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtataatttcttg ggtagtttgcagtttaaaattatgttaaaatggactatcatatgcttaccgtaactgaaagtattcgatttcttggc tttatatatcttgtggaaaggacgaggtaccgtaatttctactctgtagatgctttaaggccggtcctagcaacaag gacacctacaacaagctgataatttctactgtcgtagatgctcacctattagcgctaaggcctgtgaggtggccgc cggcatcttttttgaattcaagcttggcgtaactagatcttgagacactgctttttgcttgtactgggtctctctggttag accagatctgagcctgggagctctctggctaactagggaacccactgctaagcctcaataaagctgccttgagt gcttcaagtagtgtgtgcccgtctgttgtgtgactctggaactagagatccctcagacccttttagtcagtgtggaaa atctctagcagtacgtatagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagag tgagaggaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcattt

[0784]

[0785] ttttcactgcattctagttgtgtttgtccaaactcatcaatgtatctatcatgtctggctctagctatcccgcccctaact ccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttattatgca gaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctagggacgta cccaattcccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccct ggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcac cgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcg cgggtgtggtggtacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttccc ttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgct ttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtggccatcgccctgatagacggttttt cgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcg gtctatcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaac gcgaattttaacaaaatattaacgcttacaattaggtggcacttttcggggaaatgtgcgcggaacccctatttgttt atttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaagg aagagtatgagtattcaacatttccgtgtcgcccttattccctttttgcggcattttgccttcctgtttttgctcacccag aaacgctggtgaaagtaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaaca gcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcg cggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttga gtactcacagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatg agtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaac atgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagctgac accacgatgcctgtagcaatggcaacaacgtgcgcaaactataactggcgaactactactctagcttcccggc aacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggt ttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcatgcagcctggggccagatggtaagcc ctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagat Guide aggtgcctcactgattaagcattggtaactgtcagaccaagttactcatatatacttagattgattaaaacttcattt #030 ’ ttaatttaaaaggatctaggtgaagatcctttttataatctcatgaccaaaatcccttaacgtgagttttcgttccactga gcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaaca aaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactcttttccgaaggtaactggctt cagcagagcgcagataccaaaactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcac cgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttg gactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagc ttggagcgaacacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagg gagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccaggg ggaaacgcctggtatcttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgtcgtcagg ggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcac atgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgca gccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagccccaatacgcaaaccgcctctc cccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgca acgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtgg aattgtgagcggataacaatttcacacaggaaacgctatgaccatgattacgccaagcgcgcaattaaccctcac taaagggaacaaaagctggagctgcaagcttaatgtagtcttatgcaatactcttgtagtcttgcaacatggtaacg atgagttagcaacatgccttacaaggagagaaaaagcaccgtgcatgccgatggtggaagtaaggtggtacga tcgtgccttattaggaaggcacagacgggtctgacatggattggacgaaccactgaattgccgcattgcagagat attgtatttaagtgcctagctcgatacataaacgggtctctctggttagaccagatctgagcctgggagctctctgg ctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgt gtgatctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagg gacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggca agaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatggt gcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaa

[0786]

[0787] agaaaaaatataaataaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgtt agaaacatcagaaggctgtagacaaatactgggacagctacaaccatccctcagacaggatcagaagaactta gatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagcttta gacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacctg gaggaggagatatgagggacaattggagaagtgaattatataatataaagtagtaaaaattgaaccattaggagt agcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttcct tgggttctgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaatta Guide ttgtctggtatagtgcagcagcagac.aatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagt #030 ~ ctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctgggg atttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaa cagattggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaat tgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagttgtggaa ttggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtagtttaagaatagt ttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccg agg

[0788]

[0789] ggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagt gaacggatctcgacggtatcggttaacttttaaaagaaaaggggggattggggggtcagtgcaggggaaagaa tagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttatcgat gagtaattcatacaaaaggactcgcccctgccttggggaatcccagggaccgtcgttaaactcccactaacgtag aacccagagatcgctgcgttcccgccccctcacccgccgctctcgtcatcactgaggtggagaagagcatgcgt gaggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtc ggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcc tttttcccgagggtgggggagaaccgatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgcc gccagaacacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtgcctt gaattacttccacgcccctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagtt cgaggcttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcttgggcgctggggccgccgcg tgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctgc tgcgacgctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtattcggtttttggggcc gcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggccac cgagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatcgc cccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagtggccgcttcccggccct gctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaagga aaagggcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctcgatt agttctcaagcttttggagtacgtcgtctttaggttgggggaggggttttatgcgatggagtttccccacactgagtg ggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggatcttgg ttcattctcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtcgtgaccctagcgctacctctagag ccaccatgaccggtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggccgtacgcaccct cgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatccggaccgccacatcgagcgggtcac cgagctgcaagaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacgacggcgc Guide cgccgtggcgtctggaccacgccggagagcgtcgaagcgggggcggtgttcgccgagatcggcccgcgcat #031 ’ ggccgagttgagcggttcccggctggcc.gcgcagcaacagatggaaggcctcctggcgccgcaccggc.cca aggagcccgcgtggttcctggccaccgtcggagtctcgcccgaccaccagggcaagggtctgggcaggccgt cgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgca acctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctg gtgcatgacccgcaagcccggtgcctgaacgcgttaagtcgacaatcaactctggattacaaaatttgtgaaaga ttgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttc ccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggca acgtggcgtggtgtgcactgtgttgctgacgcaacccccactggttggggcattgccaccacctgtcagctccttt ccgggactttcgcttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacag gggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgt gttgccacctggattcgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccg cggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggcc gcctccccgcgtcgactttaagaccaatgacttacaaggcagctgtagatcttagccacttttaaaagaaaaggg ggactggaagggctaattcactcccaacgaagacaagatttcaccattatcgtttcagacccacctcccaacccc gaggggacccagagagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagata attagaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtataatttcttgggtagttt gcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatat cttgtggaaaggacgaggtaccgtaatttctactcttgtagatgctcacctattagcggctaaggcaaggacacct acaacaagctgataatttctactgtcgtagatgctttaaggccggtctagcaacctgtgaggtggccgccggcatc ttttttgaattcaagcttggcgtaactagatcttgagacactgctttttgcttgtactgggtctctctggttagaccagat ctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaa gtagtgtgtgcccgtctgttgtgtgactctggaactagagatccctcagacccttttagtcagtgtggaaaatctcta gcagtacgtatagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagag gaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcact

[0790]

[0791] gcattctagtgtgtttgtccaaactcatcaatgtatcttatcatgtctggctctagctatcccgcccctaactccgccc atcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggcc gaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctagggacgtacccaatt cccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgtt acccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcg cccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcgcgggtg tggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttc tcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacgg cacctcgaccccaaaaaacttgattagggtgatggttcacgtagtggccatcgccctgatagacggtttttcgccc tttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctat tcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaa ttttaacaaaatattaacgcttacaattaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttct aaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagt atgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgc tggtgaaagtaaagatgctgaagatcagtgggtgcacgagtgggttacatcgaactggatctcaacagcggtaa gatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtatta tcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcac agtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataa cactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatggggg atcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagctgacaccacga tgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaatta atagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctg ataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcctggggccagatggtaagccctcccgtat cgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcct Guide cactgattaagcattggtaactgtcagaccaagttactcatatatacttagattgatttaaaacttcatttttaattaa #031 ’ aaggatctaggtgaagatcctttttataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcag accccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaac caccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcag agcgcagataccaaaactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgccta catacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggtggactca agacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggag cgaacacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaa aggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaa cgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatltgtgatgtcgtcagggggg cggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttc ttcctgcgttatcccctgattctgtggataaccgtattaccgccttgagtgagctgataccgctcgccgcagccga acgaccgagcgcagcgagtcagtgagcgaggaagcggaagagccccaatacgcaaaccgcctctccccgc gcgtggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgca attaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgt gagcggataacaatttcacacaggaaacgctatgaccatgattacgccaagcgcgcaattaaccctcactaaag ggaacaaaagctggagctgcaagcttaatgtagtcttatgcaatactcttgtagtcttgcaacatggtaacgatgag ttagcaacatgccttacaaggagagaaaaagcaccgtgcatgccgattggtggaagtaaggtggtacgatcgtg ccttattaggaaggcacagacgggtctgacatggattggacgaaccactgaattgccgcattgcagagatattgt atttaagtgcctagctcgatacataaacgggtctctctggttagaccagatctgagcctgggagctctctggctaac tagggaacccactgcttaagcctcaataaagcttgcctgagtgcttcaagtagtgtgtgcccgtctgttgtgtgatc tggtaactagagatccctcagaccctttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttg aaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagagg cgaggggcggcgactggtgagtacgccaaaaatttgactagcggaggctagaaggagagagatggtgcgag agcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaa

[0792]

[0793] aaatataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgtagaaa catcagaaggctgtagacaaatactgggacagctacaaccatccctcagacaggatcagaagaacttagatcat tatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaa gatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggagga ggagatatgagggacaattggagaagtgaattatataatataaagtagtaaaaattgaaccattaggagtagcacc caccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttc Guide ttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctg #031 gtatagtgcagcagcagacaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctgggg catcaagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggg gttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaacagattg gaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaattgaaga atcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggttt aacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtagtttaagaatagtttttgct gtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgagg

[0794]

[0795] ggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagt gaacggatctcgacggtatcggttaacttttaaaagaaaaggggggattggggggtcagtgcaggggaaagaa tagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttatcgat gagtaattcatacaaaaggactcgcccctgccttggggaatcccagggaccgtcgttaaactcccactaacgtag aacccagagatcgctgcgttcccgccccctcacccgccgctctcgtcatcactgaggtggagaagagcatgcgt gaggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtc ggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcc tttttcccgagggtgggggagaaccgatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgcc gccagaacacaggtaagtgccgtgtgtggtcccgcgggcctggcctctttacgggttatggcccttgcgtgcct tgaattacttccacgcccctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagt tcgaggcttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcttgggcgctggggccgccgc gtgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctg ctgcgacgctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtattcggttttggggc cgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggcc accgagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatc gccccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagtggccgcttcccggc cctgctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaa ggaaaagggcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctc gattagttctcaagctttggagtacgtcgtctttaggttgggggaggggttttatgcgatggagtttccccacactg agtgggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggatc ttggttcattctcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtcgtgaccctagcgctacctcta gagccaccatgaccggtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggccgtacgca ccctcgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatccggaccgccacatcgagcgg gtcaccgagctgcaagaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacgacg Guide gcgccgccgtggcgtctggaccacgccggagagcgtcgaagcgggggcggtgttcgccgagatcggcccg #032 cgcatggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcctcctggcgccgcaccgg cccaaggagcccgcgtggttcctggccaccgtcggagtctcgcccgaccaccagggcaagggtctgggcag gccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgcc ccgcaacctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcg cacctggtgcatgacccgcaagcccggtgcctgaacgcgttaagtcgacaatcaactctggattacaaaatttg tgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgct attgcttcccgtatggcttcatttctcctcctgtataaatcctggttgctgtctctttatgaggagttgtggcccgttg tcaggcaacgtggcgtggtgtgcactgtgttgctgacgcaacccccactggttggggcattgccaccacctgtca gctcctttccgggactttcgctttccccctccctatgccacggcggaactcatcgccgcctgccttgcccgctgct ggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgct cgcctgtgttgccacctggatcgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggacctc cttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccct ttgggccgcctccccgcgtcgactttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaaga aaagggggactggaagggctaattcactcccaacgaagacaagatttcaccattatcgtttcagacccacctccc aaccccgaggggacccagagagggcctattcccatgattccttcatatttgcatatacgatacaaggctgttaga gagataattagaattaattgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtataatttcttg ggtagtttgcagtttaaaattatgttaaaatggactatcatatgctaccgtaacttgaaagtattcgatttcttggc tttatatatcttgtggaaaggacgaggtaccgtaatttctactcgctttaaggccggtcctagcaattgtagatcaag gacacctacaacaagctgataatttctactggctcacctattagcggctaaggtgtagatcctgtgaggtggccgc cggcatcttttttgaattcaagcttggcgtaactagatcttgagacactgctttttgcttgtactgggtctctctggttag accagatctgagcctgggagctctctggctaactagggaacccactgctaagcctcaataaagctgccttgagt gcttcaagtagtgtgtgcccgtctgttgtgtgactctggaactagagatccctcagacccttttagtcagtgtggaaa atctctagcagtacgtatagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagag tgagaggaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcattt

[0796]

[0797] ttttcactgcattctagttgtgtttgtccaaactcatcaatgtatctatcatgtctggctctagctatcccgcccctaact ccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcag aggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctagggacgtac ccaattcccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctg gcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcacc gatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcgc gggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttccct tcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctt tacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtggccatcgccctgatagacggttttt cgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcg gtctatcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaac gcgaattttaacaaaatattaacgcttacaattaggtggcacttttcggggaaatgtgcgcggaacccctatttgttt atttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaagg aagagtatgagtattcaacatttccgtgtcgcccttattcccttttgcggcattttgccttcctgtttttgctcacccag aaacgctggtgaaagtaaagatgctgaagatcagttgggtgcacgagtgggtacatcgaactggatctcaaca gcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcg cggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttga gtactcacagtcacagaaaagcatctacggatggcatgacagtaagagaattatgcagtgctgccataaccatg agtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaac atgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccata£c.aaacgacgagctgac. accacgatgcctgtagcaatggcaacaacgtgcgcaaactataactggcgaactactactctagcttcccggc aacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggt ttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcatgcagcctggggccagatggtaagcc ctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagat Guide aggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatacttagattgattaaaacttcattt #032 ttaatttaaaaggatctaggtgaagatcctttttataatctcatgaccaaaatcccttaacgtgagttttcgttccactga gcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaaca aaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactcttttccgaaggtaactggctt cagcagagcgcagataccaaaactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcac cgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttg gactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagc ttggagcgaacacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagg gagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccaggg ggaaacgcctggtatcttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgtcgtcagg ggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcac atgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgca gccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagccccaatacgcaaaccgcctctc cccgcgcgttggccgattcattaatgcagctggcacgacaggttcccgactggaaagcgggcagtgagcgca acgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtgg aattgtgagcggataacaatttcacacaggaaacgctatgaccatgattacgccaagcgcgcaattaaccctcac taaagggaacaaaagctggagctgcaagcttaatgtagtcttatgcaatactcttgtagtcttgcaacatggtaacg atgagttagcaacatgccttacaaggagagaaaaagcaccgtgcatgccgatggtggaagtaaggtggtacga tcgtgccttattaggaaggcacagacgggtctgacatggattggacgaaccactgaattgccgcattgcagagat attgtatttaagtgcctagctcgatacataaacgggtctctctggttagaccagatctgagcctgggagctctctgg ctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgt gtgatctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagg gacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggca agaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatggt gcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaa

[0798]

[0799] agaaaaaatataaataaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgtt agaaacatcagaaggctgtagacaaatactgggacagctacaaccatccctcagacaggatcagaagaactta gatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagcttta gacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacctg gaggaggagatatgagggacaattggagaagtgaattatataatataaagtagtaaaaattgaaccattaggagt agcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttcct tgggttctgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaatta Guide ttgtctggtatagtgcagcagcagac.aatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagt #032 ctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctgggg atttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaa cagattggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaat tgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattatggaattagataaatgggcaagtttgtggaa ttggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtagtttaagaatagt ttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccg agg

[0800]

[0801] ggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagt gaacggatctcgacggtatcggttaacttttaaaagaaaaggggggattggggggtcagtgcaggggaaagaa tagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttatcgat gagtaattcatacaaaaggactcgcccctgccttggggaatcccagggaccgtcgttaaactcccactaacgtag aacccagagatcgctgcgttcccgccccctcacccgccgctctcgtcatcactgaggtggagaagagcatgcgt gaggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtc ggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcc tttttcccgagggtgggggagaaccgatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgcc gccagaacacaggtaagtgccgtgtgtggtcccgcgggcctggcctctttacgggttatggcccttgcgtgcctt gaattacttccacgcccctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagtt cgaggcttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcttgggcgctggggccgccgcg tgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctgct gcgacgctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtattcggtttttggggccg cgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggccacc gagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatcgcc ccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagtggccgcttcccggccctg ctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaaggaa aagggcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctcgatta gttctcaagctttggagtacgtcgtctttaggttgggggaggggttttatgcgatggagtttccccacactgagtgg gtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggatcttggtt cattctcaagcctcagacagtggttcaaagtttttcttccatttcaggtgtcgtgaccctagcgctacctctagagc caccatgaccggtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggccgtacgcaccctc gccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatccggaccgccacatcgagcgggtcacc gagctgcaagaactctcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacgacggcgcc Guide gccgtggcgtctggaccacgccggagagcgtcgaagcgggggcggtgttcgccgagatcggcccgcgcatg #033 ’ gccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcctcctggcgccgcaccggcccaa ggagcccgcgtggttcctggccaccgtcggagtctcgcccgaccaccagggcaagggtctgggcaggccgtc gtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgcaa cctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctgg tgcatgacccgcaagcccggtgcctgaacgcgttaagtcgacaatcaactctggattacaaaattgtgaaagatt gactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttccc gtatggctttcattttctcctcctgtataaatcctggtgctgtctcttatgaggagttgtggcccgttgtcaggcaac gtggcgtggtgtgcactgtgttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttcc gggactttcgctttccccctccctatgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggg gctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgtt gccacctggattcgcgcgggacgtcctctgctacgtcccttcggccctcaatccagcggaccttccttcccgcg gcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgc ctccccgcgtcgactttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggg actggaagggctaattcactcccaacgaagacaagatttcaccattatcgtttcagacccacctcccaaccccga ggggacccagagagggcctatttcccatgattccttcatattgcatatacgatacaaggctgttagagagataatt agaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtataatttcttgggtagtttgc agttttaaaattatgttttaaaatggactatcatatgctaccgtaactgaaagtatttcgatttctggctttatatatct gtggaaaggacgaggtaccgtaatttctactcgctcacctattagcggctaaggttgtagatcaaggacacctaca acaagctgataatttctactggctttaaggccggtcctagcaatgtagatcctgtgaggtggccgccggcatctttt tgaattcaagcttggcgtaactagatcttgagacactgctttttgcttgtactgggtctctctggttagaccagatctg agcctgggagctctctggctaactagggaacccactgctaagcctcaataaagctgccttgagtgcttcaagta gtgtgtgcccgtctgttgtgtgactctggaactagagatccctcagacccttttagtcagtgtggaaaatctctagca gtacgtatagtagttcatgtcatcttattattcagtattataacttgcaaagaaatgaatatcagagagtgagaggaa cttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgca

[0802]

[0803] ttctagttgtgtttgtccaaactcatcaatgtatcttatcatgtctggctctagctatcccgcccctaactccgcccatc ccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgag gccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctagggacgtacccaattccc ctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacc caacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgccc ttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcgcgggtgtgg tggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcg ccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgattagtgctttacggcac ctcgaccccaaaaaacttgatagggtgatggtcacgtagtggccatcgccctgatagacggtttttcgccctttg acgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttt tgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaatttta acaaaatattaacgcttacaattaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaat acattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatga gtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgttttgctcacccagaaacgctggt gaaagtaaagatgctgaagatcagtgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagat ccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcc cgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcacagt cacagaaaagcatctacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacac tgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatea tgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagctgacaccacgatgcc tgtagcaatggcaacaacgttgcgcaaactataactggcgaactacttactctagcttcccggcaacaattaatag actggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataa atctggagccggtgagcgtgggtctcgcggtatcattgcagcctggggccagatggtaagccctcccgtatcgt agttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcac Guide tgattaagcatggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaattaaaag #033 ’ gatctaggtgaagatcctttttataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagacc ccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccac cgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagc gcagataccaaaactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacata cctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtctaccgggttggactcaaga cgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcga acacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaagg cggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgc ctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgtcgtcaggggggcgg agcctatggaaaaacgccagcaacgcggcctttacggttcctggccttttgctggccttttgctcacatgttctttc ctgcgttatcccctgatctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaac gaccgagcgcagcgagtcagtgagcgaggaagcggaagagccccaatacgcaaaccgcctctccccgcgc gttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaatt aatgtgagttagctcactcattaggcaccccaggcttacactttatgcttccggctcgtatgttgtgtggaattgtga gcggataacaattcacacaggaaacgctatgaccatgattacgccaagcgcgcaattaaccctcactaaaggg aacaaaagctggagctgcaagcttaatgtagtcttatgcaatactcttgtagtcttgcaacatggtaacgatgagtta gcaacatgccttacaaggagagaaaaagcaccgtgcatgccgatggtggaagtaaggtggtacgatcgtgcc ttattaggaaggcacagacgggtctgacatggattggacgaaccactgaattgccgcattgcagagatattgtatt taagtgcctagctcgatacataaacgggtctctctggttagaccagatctgagcctgggagctctctggctaacta gggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgatct ggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttga aagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggc gaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatggtgcgaga gcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaa

[0804]

[0805] aatataaataaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaac atcagaaggctgtagacaaatactgggacagctacaaccatccctcagacaggatcagaagaacttagatcata tataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaag atagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggaggag gagatatgagggacaattggagaagtgaattatataatataaagtagtaaaaattgaaccattaggagtagcaccc accaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttctt Guide gggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggt #033 atagtgcagcagcagacaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggc atcaagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttgggg ttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaacagattgg aatcacacgacctggatggagtgggacagagaaattaacaattacacaagctaatacactccttaattgaagaat cgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaa cataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtagtttaagaatagtttttgctgt actttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgagg

[0806]

[0807] ggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagt gaacggatctcgacggtatcggttaacttttaaaagaaaaggggggattggggggtcagtgcaggggaaagaa tagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttatcgat gagtaattcatacaaaaggactcgcccctgccttggggaatcccagggaccgtcgttaaactcccactaacgtag aacccagagatcgctgcgttcccgccccctcacccgccgctctcgtcatcactgaggtggagaagagcatgcgt gaggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtc ggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcc tttttcccgagggtgggggagaaccgatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgcc gccagaacacaggtaagtgccgtgtgtggtcccgcgggcctggcctctttacgggttatggcccttgcgtgcct tgaattacttccacgcccctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagt tcgaggcttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcttgggcgctggggccgccgc gtgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctg ctgcgacgctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtattcggttttggggc cgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggcc accgagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatc gccccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagtggccgcttcccggc cctgctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaa ggaaaagggcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctc gattagttctcaagctttggagtacgtcgtctttaggttgggggaggggttttatgcgatggagtttccccacactg agtgggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggatc ttggttcattctcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtcgtgaccctagcgctacctcta gagccaccatgaccggtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggccgtacgca ccctcgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatccggaccgccacatcgagcgg gtcaccgagctgcaagaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacgacg Guide gcgccgccgtggcgtctggaccacgccggagagcgtcgaagcgggggcggtgttcgccgagatcggcccg #034 ’ cgcatggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcctcctggcgccgcaccgg cccaaggagcccgcgtggttcctggccaccgtcggagtctcgcccgaccaccagggcaagggtctgggcag gccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgcc ccgcaacctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcg cacctggtgcatgacccgcaagcccggtgcctgaacgcgttaagtcgacaatcaactctggattacaaaatttg tgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgct attgcttcccgtatggcttcatttctcctcctgtataaatcctggttgctgtctctttatgaggagttgtggcccgttg tcaggcaacgtggcgtggtgtgcactgtgttgctgacgcaacccccactggttggggcattgccaccacctgtca gctcctttccgggactttcgctttccccctccctatgccacggcggaactcatcgccgcctgccttgcccgctgct ggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgct cgcctgtgttgccacctggatcgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggacctc cttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccct ttgggccgcctccccgcgtcgactttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaaga aaagggggactggaagggctaattcactcccaacgaagacaagatttcaccattatcgtttcagacccacctccc aaccccgaggggacccagagagggcctattcccatgattccttcatatttgcatatacgatacaaggctgttaga gagataattagaattaattgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtataatttcttg ggtagtttgcagtttaaaattatgttaaaatggactatcatatgctaccgtaacttgaaagtattcgatttcttggc tttatatatcttgtggaaaggacgaggtaccgtaatttctactcttgtagatataccaaactgtccccagaacagtaat ttctactgtcgtagattcccaatctgtgtagttagcagcttttttgaattcaacttggcgtaactagatcttgagacact gctttttgcttgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccac tgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactaga gatccctcagaccctttagtcagtgtggaaaatctctacagtacgtatagtagttcatgtcatcttattattcagtattt ataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttattgcagcttataatggttacaaataaagca atagcatcacaaattcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatctta

[0808]

[0809] tcatgtctggctctagctacccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctcc gccccatggctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagt gaggaggcttttttggaggcctagggacgtacccaattcgccctatagtgagtcgtattacgcgcgctcactggc cgtcgtttacaagtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgc cagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgg gacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgcca ggccctagcgcccgctcctttcgctttcttcccttccttctcgccacgttcgccggctttccccgtcaagctctaaat cgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttc acgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtcacgttctttaatagtggactcttgt tccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattgg ttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcactttt cggggaaatgtgcgcggaacccctatttgtttattttctaaatacattcaaatatgtatccgctcatgagacaataac cctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattccctttttt gcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgc acgagtgggttacatcgaactgatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaa tgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcg ccgcatacactattctcagaatgacttggtgagtactcaccagtcacagaaaagcatcttacggatggcatgaca gtaagagaattagcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggag gaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagc tgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaaca ttaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggac cacttctgcgctcggcccttccggctggctggttattgctgataaatctggagccggtgagcgtgggtctcgcgg tatcatgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacacggggagtcaggcaact atggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagttt Guide actcatatatactttagattgattaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatg #034 ’ accaaaatcccttaacgtgagttttcgttccactagcgtcagaccccgtagaaaagatcaaaggatcttcttgagat cctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaa gagctaccaactctttttccgaaggtaactggctcagcagagcgcagataccaaatactgttcttctagtgtagcc gtagttaggccaccacttcaagaacctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgc tgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgg gctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagc gtgagctatgagaaagcccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcg gaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccac ctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcgcct ttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtat accgccttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaa gcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgagctggcacgacag gtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccagg ctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatg accatgattacgccaagcgcgcaattaaccctcactaaaggaacaaaagctggagctgcaagcttaatgtagtct tatgcaatactcttgtagtcttgcaacatggtaacgatgagttagcaacatgccttacaaggagagaaaaagcacc gtgcatgccgatggtggaagtaaggtggtacgatcgtgccttattaggaaggcaacagacgggtctgacatgg attggacgaaccactgaattgccgcattcagagatatgtatttaagtgcctagctcgatacataaacgggtctctct ggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgc cttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagt gtggaaatctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgac gcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaatt ttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcg cgtgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagg

[0810]

[0811] gagctagaacgattcgcagttaatcctggcctgtagaaacatcagaaggctgtagacaaatactgggacagcta caaccatcccttcagacaggatcagaagaacttagatcattatataatacagtagaaccctctattgtgtgcatcaa aggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagaccacc gcacagcaagcggccgctgatcttcagacctggaggaggagatatgagggacaattggagaagtgaattatata aatataaagtagtaaaaattgaaccattaggagtagcacccacaaggcaaagagaagagtggtgcagagagaa aaaagagcagtgggaataggagcttgttcctgggttcttgggagcagcaggaagcactatgggcgcagcgtc Guide aatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgagggctatt #034 gaggcgcaacagcatctgttgcaaccacagtctggggcatcaagcagctccaggcaagaatcctggctgtgga aagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgcctt ggaatgctagttggagtaataaatctctggaacagattggaatcacacgacctggatggagtgggacagagaa attaacaatacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaatt attggaattagataaatgggcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataa tgatagtaggaggcttggtaggtttaagaatagttttgctgtactttctatagtgaatagagttagcagggatattca ccattatcgtttcagacccacctcccaaccccgagg

[0812]

[0813] ggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagt gaacggatctcgacggtatcggttaacttttaaaagaaaaggggggattggggggtcagtgcaggggaaagaa tagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttatcgat gagtaattcatacaaaaggactcgcccctgccttggggaatcccagggaccgtcgttaaactcccactaacgtag aacccagagatcgctgcgttcccgccccctcacccgccgctctcgtcatcactgaggtggagaagagcatgcgt gaggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtc ggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcc tttttcccgagggtgggggagaaccgatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgcc gccagaacacaggtaagtgccgtgtgtggtcccgcgggcctggcctctttacgggttatggcccttgcgtgcctt gaattacttccacgcccctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagtt cgaggcttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcttgggcgctggggccgccgcg tgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctgc tgcgacgctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtattcggtttttggggcc gcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggccac cgagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatcgc cccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagtggccgcttcccggccct gctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaagga aaagggcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctcgatt agttctcaagcttttggagtacgtcgtctttaggttgggggaggggtttatgcgatggagtttccccacactgagtg ggtggagactgaagttaggccagctggcacttgatgtaattctccttggaatttgccctttttgagtttggatcttggt tcattctcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtcgtgaccctagcgctacctctagag ccaccatgaccggtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggccgtacgcaccct cgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatccggaccgccacategagcgggtcac cgagctgcaagaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacgacggcgc Guide_ cgccgtggcgtctggaccacgccggagagcgtcgaagcgggggcggtgttcgccgagatcggcccgcgcat #035 ggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcctcctggcgccgcaccggccca aggagcccgcgtggttcctggccaccgtcggagtctcgcccgaccaccagggcaagggtctgggcaggccgt cgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgca acctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctg gtgcatgacccgcaagcccggtgcctgaacgcgttaagtcgacaatcaactctggattacaaaatttgtgaaaga ttgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttc ccgtatggctttcattttctcctccttgtataaatcctggtgctgtctctttatgaggagttgtggcccgttgtcaggca acgtggcgtggtgtgcactgtgttgctgacgcaacccccactggttggggcattgccaccacctgtcagctccttt ccgggactttcgcttccccctccctattgccacggcggaactcatcgccgcctgcctgcccgctgctggacag gggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgt gttgccacctggattcgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccg cggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggcc gcctccccgcgtcgactttaagaccaatgacttacaaggcagctgtagatcttagccactttaaaagaaaaggg ggactggaagggctaattcactcccaacgaagacaagatttcaccattatcgtttcagacccacctcccaacccc gaggggacccagagagggcctattcccatgattccttcatattgcatatacgatacaaggctgttagagagata attagaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtataatttcttgggtagttt gcagttttaaaatatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttctggctttatatat cttgtggaaaggacgaggtaccgtaatttctactcttgtagattcccaatctgtgtagttagcagctaatttctactgtc gtagatataccaaactgtccccagaacagtttlttgaattcaacttggcgtaactagatcttgagacactgctttttgct tgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagc ctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctca gacccttttagtcagtgtggaaaatctctacagtacgtatagtagttcatgtcatcttattattcagtatttataacttgca aagaaatgaatatcagagagtgagaggaacttgtttattgcagcttataatggttacaaataaagcaatagcatcac aaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgg

[0814]

[0815] ctctagctacccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatgg ctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggctt ttttggaggcctagggacgtacccaattcgccctatagtgagtcgtattacgcgcgctcactggccgtcgtttaca agtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgta atagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccct gtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccaggccctagcg cccgctcctttcgctttcttoccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctcc ctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtggg ccatcgccctgatagacggtttttcgccctttgacgttggagtcacgttctttaatagtggactctgttccaaactgg aacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatg agctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaat gtgcgcggaacccctatttgtttattttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaat gcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcatttg ccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggt tacatcgaactgatctcaacagcggtaagatccttgagagtttcgccccgaagaacgttttccaatgatgagcact tttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacact attctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaatt agcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaagga gctaaccgcttttttgcacaacatgggggatcatgtaactcgcctgatcgttgggaaccggagctgaatgaagcc ataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaacattaactggcga actacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgc tc.ggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcag cactggggccagatggtaagccctcccgtatcgtagttatctacacacggggagtcaggcaactatggatgaac gaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatata Guide_ ctttagattgattaaaacttcattttaattaaaaggatctaggtgaagatcctttgataatctcatgaccaaaatcc #035 cttaacgtgagttttcgttccactagc.gtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttc.tg cgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctacca actctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggc caccacttcaagaacctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtgg cgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacgg ggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatg agaaagcccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggag agcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttg agcgtcgattttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcgcctttttacggtt cctggcctttgctggccttttgctcacatgttctttcctgcgtatcccctgattctgtggataaccgtataccgccttt gagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaag agcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgagctggcacgacaggtttcccg actggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacact ttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgatt acgccaagcgcgcaattaaccctcactaaaggaacaaaagctggagctgcaagcttaatgtagtcttatgcaata ctcttgtagtcttgcaacatggtaacgatgagttagcaacatgccttacaaggagagaaaaagcaccgtgcatgc cgattggtggaagtaaggtggtacgatcgtgccttattaggaaggcaacagacgggtctgacatggattggacg aaccactgaattgccgcattcagagatattgtatttaagtgcctagctcgatacataaacgggtctctctggtagac cagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgcctgagtgc ttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaat ctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggact cggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaatttgactagc ggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgtgggaa aaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctag

[0816]

[0817] aacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccat cccttcagacaggatcagaagaacttagatcattatataatacagtagaaccctctattgtgtgcatcaaaggatag agataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacag caagcggccgctgatcttcagacctggaggaggagatatgagggacaattggagaagtgaattatataaatata aagtagtaaaaattgaaccattaggagtagcacccacaaggcaaagagaagagtggtgcagagagaaaaaag agcagtgggaataggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatga Guide cgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgagggctattgagg #035 cgcaacagcatctgttgcaaccacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagat acctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaat gctagttggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaac aatacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattgga attagataaatgggcaagttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatag taggaggcttggtaggtttaagaatagtttttgctgtactttctatagtgaatagagttagcagggatattcaccattat cgtttcagacccacctcccaaccccgagg

[0818]

[0819] ggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagt gaacggatctcgacggtatcggttaacttttaaaagaaaaggggggattggggggtcagtgcaggggaaagaa tagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttatcgat gagtaattcatacaaaaggactcgcccctgccttggggaatcccagggaccgtcgttaaactcccactaacgtag aacccagagatcgctgcgttcccgccccctcacccgccgctctcgtcatcactgaggtggagaagagcatgcgt gaggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtc ggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcc tttttcccgagggtgggggagaaccgatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgcc gccagaacacaggtaagtgccgtgtgtggtcccgcgggcctggcctctttacgggtatggcccttgcgtgcct tgaattacttccacgcccctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagt tcgaggcttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcttgggcgctggggccgccgc gtgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctg ctgcgacgctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtattcggttttggggc cgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggcc accgagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatc gccccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagtggccgcttcccggc cctgctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaa ggaaaagggcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctc gattagttctcaagctttggagtacgtcgtctttaggttgggggaggggttttatgcgatggagtttccccacactg agtgggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggatc ttggttcattctcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtcgtgaccctagcgctacctcta gagccaccatgaccggtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggccgtacgca ccctcgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatccggaccgccacatcgagcgg gtcaccgagctgcaagaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacgacg Guide_ gcgccgccgtggcgtctggaccacgccggagagcgtcgaagcgggggcggtgttcgccgagatcggcccg #036 cgcatggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcctcctggcgccgcaccgg cccaaggagcccgcgtggttcctggccaccgtcggagtctcgcccgaccaccagggcaagggtctgggcag gccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgcc ccgcaacctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcg cacctggtgcatgacccgcaagcccggtgcctgaacgcgttaagtcgacaatcaactctggattacaaaatttg tgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgct attgcttcccgtatggctttcattttctcctcctgtataaatcctggttgctgtctctttatgaggagttgtggcccgttg tcaggcaacgtggcgtggtgtgcactgtgttgctgacgcaacccccactggttggggcattgccaccacctgtca gctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgct ggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgct cgcctgtgttgccacctggatcgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggacctc cttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccct ttgggccgcctccccgcgtcgactttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaaga aaagggggactggaagggctaattcactcccaacgaagacaagatttcaccattatcgtttcagacccacctccc aaccccgaggggacccagagagggcctattcccatgattccttcatatttgcatatacgatacaaggctgtaga gagataattagaattaattgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtataatttcttgg gtagtttgcagtttaaaattatgtttaaaatggactatcatatgcttaccgtaactgaaagtatttcgatttcttggctt tatatatcttgtggaaaggacgaggtaccgtaatttctactcttgtagatataccaaactgtccccagaacagtaattt ctactgtcgtagattcccaatctgtgtagttagcagcaaatttctactctatagatttttttgaattcaagctlggcgtaa ctagatcttgagacactgcttttgcttgtactgggtctctctggttagaccagatctgagcctgggagctctctggct aactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgt gactctggtaactagagatccctcagacccttttagcagtgtggaaaatctctagcagtacgtatagtagttcatgtc atcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttattgcagcttataat ggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaa

[0820]

[0821] actcatcaatgtatctttcatgtctggctctagctatcccgcccctaactccgcccatcccgcccctaactccgccca gttccgcccattctccgccccatggctgactaatttttttatttatgcagaggccgaggccgcctcggcctctgagc tattccagaagtagtgaggaggcttttttggaggcctagggacgtacccaattcgccctatagtgagtcgtattacg cgcgtcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagca catccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcct gaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtacc gctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttoccc gtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgat tagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccttgacgttggagtccacgttcttta atagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccg atttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaat ttaggtggcacttttcggggaaatgtgcgcgaacccctatttgtttatttttctaaatacattcaaatatgtatccgctca tgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcc cttattcccttttttgcggcattttgccttcctgttttgctcacccagaaacgctggtgaaagtaaaagatgctgaaga tcagttgggtgcacggtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaa gaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaaga gcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacg gatggatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgac aacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttg ggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcacaacg ttgcgcaaactattaactggcgaactactactctagcttcccggcaacaattaatagactggatggaggcggata aagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcg tgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgatcgtagttatctacacgacgggg agtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactg Guide_ tcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttt #036 tgataatctcatgaccaaaatcccttaagtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaag gatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttg tttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttc ttctagtgtagccgtagttagccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgtt accagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggc gcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactga gatacctacacgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaag cggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtc gggtttcgccacctctgacttgagcgtcgattttgtgatgctcgtcaggggggcggagcctatggaaaacgcca gcaacgcggcctttttacggttcctggccttttgctggcctttgctcacatgttctttcctgcgttatcccctgattctg tggataaccgtataccgccttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtc agtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcttggccgattcattaatgca gctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcat taggcaccccaggcttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacaca ggaaacagctatgaccatgattacgccaagcggcaattaaccctcactaaagggaacaaaagctggagctgca agcttaatgtagtcttatgcaatactcttgtagtcttgcaacatggtaacgatgagttagcaacatgccttacaagga gagaaaaagcaccgtgcatgccgattggtggaagtaaggtggtacgatcgtgccttataggaaggcaacaga cgggtctgacatggattggacgaacactgaattgccgcattgcagagatatgtatttaagtgcctagctcgataca taaacgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcc tcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcag acccttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaaccaga ggagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtga gtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggg agaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagt

[0822]

[0823] atgggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaa tactgggacagctacaaccatcccttcagacaggatcagaagaacttagatcatatataatacagtagcaaccctc tattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaaca aaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggaggaggagatatgagggacaattgg agaagtgaatatataaatataaagtagtaaaaatgaaccataggagtagcacccaccaaggcaaagagaaga gtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagcagcaggaagca Guide ctatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaac #036 aatttgctgagggctattgaggcgcacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaa gaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttg caccactgctgtgccttggaatgctagttggagtaataaatctctggaacagatttggaatcacacgacctggatg gagtgggcagagaaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaa agaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaattggctgtggt atataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtactttcatagtgaatagagt taggcagggatattcaccattatcgtttcagacccacctcccaaccccgagg

[0824]

[0825] ggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagt gaacggatctcgacggtatcggttaacttttaaaagaaaaggggggattggggggtcagtgcaggggaaagaa tagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttatcgat gagtaattcatacaaaaggactcgcccctgccttggggaatcccagggaccgtcgttaaactcccactaacgtag aacccagagatcgctgcgttcccgccccctcacccgccgctctcgtcatcactgaggtggagaagagcatgcgt gaggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtc ggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcc tttttcccgagggtgggggagaaccgatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgcc gccagaacacaggtaagtgccgtgtgtggtcccgcgggcctggcctctttacgggtatggcccttgcgtgcct tgaattacttccacgcccctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagt tcgaggcttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcttgggcgctggggccgccgc gtgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctg ctgcgacgctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtattcggttttggggc cgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggcca ccgagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatcg ccccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagtggccgcttcccggcc ctgctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaag gaaaagggcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctcg attagtctcaagcttttggagtacgtcgtctttaggttgggggaggggttttatgcgatggagtttccccacactga gtgggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggatctt ggttcattctcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtcgtgaccctagcgctacctctag agccaccatgaccggtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggccgtacgcac cctcgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatccggaccgccacatcgagcgggt caccgagctgcaagaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacgacgg Guide_ cgccgccgtggcgtctggaccacgccggagagcgtcgaagcgggggcggtgttcgccgagatcggcccgc #037 gcatggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcctcctggcgccgcaccgg cccaaggagcccgcgtggttcctggccaccgtcggagtctcgcccgaccaccagggcaagggtctgggcag gccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgcc ccgcaacctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcg cacctggtgcatgacccgcaagcccggtgcctgaacgcgttaagtcgacaatcaactctggattacaaaatttgt gaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgcta ttgctcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgt caggcaacgtggcgtggtgtgcactgtgttgctgacgcaacccccactggttggggcattgccaccacctgtca gctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgct ggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgct cgcctgtgttgccacctggatcgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggacctc cttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccct ttgggccgcctccccgcgtcgactttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaaga aaagggggactggaagggctaattcactcccaacgaagacaagatttcaccattatcgtttcagacccacctccc aaccccgaggggacccagagagggcctattcccatgattccttcatatttgcatatacgatacaaggctgtaga gagataattagaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtataatttcttgg gtagtttgcagtttaaaattatgtttaaaatggactatcatatgcttaccgtaactgaaagtatttcgatttcttggctt tatatatcttgtggaaaggacgaggtaccgtaatttctactcttgtagatataccaaactgtccccagaacaggcttt aaggccggtcctagcaataatttctactgtcgtagattcccaatctgtgtagtagcagcgctcacctattagcggct aaggttttttgaattcaagcttggcgtaactagatcttgagacactgctttttgcttgtactgggtctctctggttagac cagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgc ttcaagtagtgtgtgcccgtctgttgtgtgactctggtactagagatccctcagacccttttagtcagtgtggaaaat ctctagcagtacgtatagtagttcatgtcatctatlattcagtatttataacttgcaaagaaatgaatatcagagagtg agaggaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcattttt

[0826]

[0827] ttcactgcattctagttgtggttgtccaaactcatcaatgtatcttatcatgtctggctctagctatcccgcccctaactc cgcccatcccgcccctaactccgcc.cagttccgccc.attctccgccccatggctgactaattttttttatttatgcag aggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctagggacgtac ccaattcgcctatagtgagtcgtatacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccct ggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcac cgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcg cgggtgtggtggtacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttccc ttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgct ttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggcatcgccctgatagacggttttt cgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcg gtctatcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaac gcgaattttaacaaaatattaacgcttacaatttagtggcacttttcggggaaatgtgcgcggaacccctatttgttta ttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaagga agagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccaga aacgctggtgaaagtaaaaatgctgaagatcagttgggtgcacgagtgggtacatcgaactggatctcaacag cggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgc ggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgag tactcaccgtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatga gtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaaca tgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtaca ccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggca acaattaatagactggatggaggcggataaagtgcaggaccacttctgcgctcggcccttccggctggctggtt tattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcacggggccagatggtaagcc ctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagat Guide_ aggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatacttagattgattaaaacttcattt #037 ttaatttaaaaggatctaggtgaagatcctttttgtaatctcatgaccaaaatcccttaacgtgagttttcgttccactg agcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaac aaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggc ttcagcagagcgcagataccaaatatgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagca ccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggtt ggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccag cttggagcgaacgactacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaag ggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagg gggaaacgcctggtatcttatagtcctgtcgggtttcgccacctctgacttgagcgtcgattttgtgatgctgtcag gggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggcctttgctca catgttctttcctgcgttatcccctgatctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgc agccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgccaatacgcaaaccgcctct ccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgc aacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtg gaatgtgagcggataacaattcacacaggaaacagtatgaccatgatacgccaagcgcgcaattaaccctca ctaaagggaacaaaagctggagctgcaagcttaatgtagtcttatgcaatactcttgtagtcttgcaacatggtaac gatgagttagcaacatgccttacaaggagagaaaaagcaccgtgcatgccgatggtggaagtaaggtggtacg atcgtgccttattaggaaggcaacgacgggtctgacatggattggacgaaccactgaattgccgcattgcagaga tattgtattaagtgcctagctcgatacataaacgggtctctctggttagaccagatctgagcctgggagctctctgg ctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtg tgacttggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacaggga cttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaag aggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggg cgagagcgtcagtataagcgggggagaattagatcgcgatgggaaaaaattcggtaaggccagggggaaa

[0828]

[0829] no gaaaaaatataaataaaacatatagtatgggcaagcagggagctagaacgattcgcagtaatcctggcctgtta gaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcgacaggatcagaagaacttaga tcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttaga caagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggag gaggagatatgagggacaattggagaagtgaattatataatataaagtagtaaaaattgaaccattaggagtagca cccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggt Guide tcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtct #037 ggtatagtgcagcagcagaacatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggg gcatcaagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttgg ggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaacagatt tggatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaatgaaga atcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggttt aacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggttaagaatagtttttgc tgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgagg

[0830]

[0831] ggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagt gaacggatctcgacggtatcggttaacttttaaaagaaaaggggggattggggggtcagtgcaggggaaagaa tagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttatcgat gagtaattcatacaaaaggactcgcccctgccttggggaatcccagggaccgtcgttaaactcccactaacgtag aacccagagatcgctgcgttcccgccccctcacccgccgctctcgtcatcactgaggtggagaagagcatgcgt gaggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtc ggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcc tttttcccgagggtgggggagaaccgatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgcc gccagaacacaggtaagtgccgtgtgtggtcccgcgggcctggcctctttacgggttatggcccttgcgtgcctt gaattacttccacgcccctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagtt cgaggcttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcttgggcgctggggccgccgcg tgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgac.ctgct gcgacgctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtattcggtttttggggccg cgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggccacc gagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatcgcc ccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagtggccgcttcccggccctg ctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaaggaa aagggcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctcgatta gttctcaagctttggagtacgtcgtctttaggttgggggaggggttttatgcgatggagtttccccacactgagtgg gtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggatcttggtt cattctcaagcctcagacagtggttcaaagtttttcttccatttcaggtgtcgtgaccctagcgctacctctagagc caccatgaccggtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggccgtacgcaccctc gccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatccggaccgccacatcgagcgggtcacc gagctgcaagaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacgacggcgcc Guide_ gccgtggcgtctggaccacgccggagagcgtcgaagcgggggcggtgttcgccgagatcggcccgcgcatg #038 gccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcctcctggcgccgcaccggcccaa ggagcccgcgtggttcctggccaccgtcggagtctcgcccgaccaccagggcaagggtctgggcaggccgtc gtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgcaa cctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctgg tgcatgacccgcaagcccggtgcctgaacgcgttaagtcgacaatcaactctggattacaaaatttgtgaaagatt gactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcc cgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaa cgtggcgtggtgtgcactgtgttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttc cgggacttcgctttccccctccctattgccacggcggaactcatcgccgcctgcctgcccgctgctggacagg ggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtg tgccacctggattcgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgc ggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccg cctccccgcgtcgactttaagaccaatgacttacaaggcagctgtagatcttagccacttttaaaagaaaagggg gactggaagggctaattcactcccaacgaagacaagatttcaccattatcgtttcagacccacctcccaaccccg aggggacccagagagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataat tagaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtataatttcttgggtagtttg cagtttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtattcgatttcttggctttatatatct tgtggaaaggacgaggtaccgtaatttctactcttgtagatataccaaactgtccccagaacaggctcacctattag cggctaaggtaattctactgtcgtagattcccaatctgtgtagtagcagcgcttaaggccggtcctagcaatttttt gaattcaagcttggcgtaactagatcttgagacactgctttttgcttgtactgggtctctctggttagaccagatctga gcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgctcaagtag tgtgtgcccgtctgttgtgtgactctggtactagagatccctcagacccttttagtcagtgtggaaaatctctagcag tacgtatagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaac ttgtttattgcagcttataatggtacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcat

[0832]

[0833] tctagtgtggttgtccaaactcatcaatgtatctatcatgtctggctctagctatcccgcccctaactccgcccatc ccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgag gccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctagggacgtacccaattcgc ctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacc caacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgccc ttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcgcgggtgtgg tggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcg ccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcac ctcgaccccaaaaaacttgatagggtgatggttcacgtagtgggcatcgccctgatagacggtttttcgccctttg acgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttt tgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaatttta acaaaatattaacgcttacaatttagtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaat acattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatga gtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgttttgctcacccagaaacgctggt gaaagtaaaaatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatc cttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatccc gtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccgtc acagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacact gcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcat gtaactcgcctgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtacaccacgatgcct gtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatag actggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataa atctggagccggtgagcgtgggtctcgcggtatcattgcagcacggggccagatggtaagccctcccgtatcgt agttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcac Guide_ tgattaagcatggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaag #038 gatctaggtgaagatcctttttgtaatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagacc ccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccac cgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagc gcagataccaaatatgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacata cctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtctaccgggttggactcaagac gatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaa cgactacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggc ggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcc tggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctgtcaggggggcgga gcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcc tgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacg accgagcgcagcgagtcagtgagcgaggaagcggaagagcgccaatacgcaaaccgcctctccccgcgcg ttggccgattcataatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaatta atgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgag cggataacaatttcacacaggaaacagtatgaccatgattacgccaagcgcgcaattaaccctcactaaaggga acaaaagctggagctgcaagcttaatgtagtcttatgcaatactcttgtagtcttgcaacatggtaacgatgagttag caacatgccttacaaggagagaaaaagcaccgtgcatgccgattggtggaagtaaggtggtacgatcgtgcctt attaggaaggcaacgacgggtctgacatggattggacgaaccactgaattgccgcattgcagagatattgtattta agtgcctagctcgatacataaacgggtctctctggttagaccagatctgagcctgggagctctctggctaactagg gaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgacttggt aactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaag cgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgag gggcggcgactggtgagtacgccaaaaattgactagcggaggctagaaggagagagatggggcgagagc gtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaat

[0834]

[0835] ataaattaaaacatatagtatgggcaagcagggagctagaacgatcgcagttaatcctggcctgttagaaacatc agaaggctgtagacaaatactgggacagctacaaccatcccttcgacaggatcagaagaacttagatcattatat aatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagata gaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggaggaggag atatgagggacaattggagaagtgaattatataatataaagtagtaaaaattgaaccattaggagtagcacccacc aaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgg Guide gagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtata #038 gtgcagcagcagaacatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatca agcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgc tctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaacagatttggatc acacgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatcgc aaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacat aacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggttaagaatagtttttgctgtact ttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgagg

[0836]

[0837] ggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagt gaacggatctcgacggtatcggttaacttttaaaagaaaaggggggattggggggtcagtgcaggggaaagaa tagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttatcgat gagtaattcatacaaaaggactcgcccctgccttggggaatcccagggaccgtcgttaaactcccactaacgtag aacccagagatcgctgcgttcccgccccctcacccgccgctctcgtcatcactgaggtggagaagagcatgcgt gaggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtc ggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcc tttttcccgagggtgggggagaaccgatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgcc gccagaacacaggtaagtgccgtgtgtggtcccgcgggcctggcctctttacgggttatggcccttgcgtgcctt gaattacttccacgcccctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagtt cgaggcttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcttgggcgctggggccgccgcg tgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgac.ctgct gcgacgctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtattcggtttttggggccg cgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggccacc gagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatcgcc ccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagtggccgcttcccggccct gctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaagga aaagggcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctcgatt agttctcaagcttttggagtacgtcgtctttaggttgggggaggggtttatgcgatggagtttccccacactgagtg ggtggagactgaagttaggccagctggcacttgatgtaattctccttggaatttgccctttttgagtttggatcttggt tcattctcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtcgtgaccctagcgctacctctagag ccaccatgaccggtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggcc.gtac.gcaccct cgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatccggaccgccacategagcgggtcac cgagctgcaagaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacgacggcgc Guide_ cgccgtggcgtctggaccacgccggagagcgtcgaagcgggggcggtgttcgccgagatcggcccgcgcat #039 ggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcctcctggcgccgcaccggccca aggagcccgcgtggttcctggccaccgtcggagtctcgcccgaccaccagggcaagggtctgggcaggccgt cgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgca acctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctg gtgcatgacccgcaagcccggtgcctgaacgcgttaagtcgacaatcaactctggattacaaaatttgtgaaaga ttgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttc ccgtatggctttcattttctcctccttgtataaatcctggtgctgtctctttatgaggagttgtggcccgttgtcaggca acgtggcgtggtgtgcactgtgttgctgacgcaacccccactggttggggcattgccaccacctgtcagctccttt ccgggactttcgcttccccctccctattgccacggcggaactcatcgccgcctgcctgcccgctgctggacag gggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgt gttgccacctggattcgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccg cggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggcc gcctccccgcgtcgactttaagaccaatgacttacaaggcagctgtagatcttagccactttaaaagaaaaggg ggactggaagggctaattcactcccaacgaagacaagatttcaccattatcgtttcagacccacctcccaacccc gaggggacccagagagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagata attagaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtataatttcttgggtagttt gcagttttaaaatatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttctggctttatatat cttgtggaaaggacgaggtaccgtaatttctactcttgtagatgctttaaggccggtcctagcaaataccaaactgt ccccagaacagtaatttctactgtcgtagatgctcacctattagcgctaaggtcccaatctgtgtagttagcagcttt ttgaattcaagcttggcgtaactagatcttgagacactgctttttgcttgtactgggtctctctggttagaccagatctg agcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagta gtgtgtgcccgtctgttgtgtgactctggtactagagatccctcagacccttttagtcagtgtggaaaatctctagca gtacgtatagtagttcatgtcatcttattattcagtattataacttgcaaagaaatgaatatcagagagtgagaggaa cttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgca

[0838]

[0839] ttctagttgtggttgtccaaactcatcaatgtatcttatcatgtctggctctagctatcccgcccctaactccgcccatc ccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgag gccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctagggacgtacccaattcgc ctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacc caacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgccc ttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcgcgggtgtgg tggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcg ccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcac ctcgaccccaaaaaacttgatagggtgatggttcacgtagtgggcatcgccctgatagacggtttttcgccctttg acgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttt tgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaatttta acaaaatattaacgcttacaatttagtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaat acattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatga gtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgttttgctcacccagaaacgctggt gaaagtaaaaatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatc cttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatccc gtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccgtc acagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacact gcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcat gtaactcgcctgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtacaccacgatgcct gtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatag actggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataa atctggagccggtgagcgtgggtctcgcggtatcattgcagcacggggccagatggtaagccctcccgtatcgt agttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcac Guide_ tgattaagcatggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaattaaaag #039 gatctaggtgaagatcctttttgtaatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagacc ccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccac cgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagc gcagataccaaatatgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacata cctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtctaccgggttggactcaaga cgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcga acgactacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaagg cggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgc ctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctgtcaggggggcgg agcctatggaaaaacgccagcaacgcggcctttacggttcctggccttttgctggccttttgctcacatgttctttc ctgcgttatcccctgatctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaac gaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgccaatacgcaaaccgcctctccccgcgc gttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaatt aatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtga gcggataacaatttcacacaggaaacagtatgaccatgattacgccaagcgcgcaattaaccctcactaaaggg aacaaaagctggagctgcaagcttaatgtagtcttatgcaatactcttgtagtcttgcaacatggtaacgatgagtta gcaacatgccttacaaggagagaaaaagcaccgtgcatgccgattggtggaagtaaggtggtacgatcgtgcct tattaggaaggcaacgacgggtctgacatggattggacgaaccactgaattgccgcattgcagagatattgtattt aagtgcctagctcgatacataaacgggtctctctggttagaccagatctgagcctgggagctctctggctaactag ggaacccactgctaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgacttgg taactagagatccctcagaccctttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaa gcgaaagggaaaccagaggagctctctcgacgcaggactcggctgctgaagcgcgcacggcaagaggcga ggggcggcgactggtgagtacgccaaaaatttgactagcggaggctagaaggagagagatggggcgagag cgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaa

[0840]

[0841] tataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacat cagaaggctgtagacaaatactgggacagctacaaccatcccttcgacaggatcagaagaacttagatcattata taatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagat agaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggaggagga gatatgagggacaattggagaagtgaattatataatataaagtagtaaaaattgaaccattaggagtagcacccac caaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgg Guide gagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtata #039 gtgcagcagcagaacatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatca agcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgc tctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaacagatttggatc acacgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatcgc aaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacat aacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggttaagaatagtttttgctgtact ttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgagg

[0842]

[0843] ggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagt gaacggatctcgacggtatcggttaacttttaaaagaaaaggggggattggggggtcagtgcaggggaaagaa tagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttatcgat gagtaattcatacaaaaggactcgcccctgccttggggaatcccagggaccgtcgttaaactcccactaacgtag aacccagagatcgctgcgttcccgccccctcacccgccgctctcgtcatcactgaggtggagaagagcatgcgt gaggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtc ggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcc tttttcccgagggtgggggagaaccgatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgcc gccagaacacaggtaagtgccgtgtgtggtcccgcgggcctggcctctttacgggttatggcccttgcgtgcctt gaattacttccacgcccctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagtt cgaggcttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcttgggcgctggggccgccgcg tgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgac.ctgct gcgacgctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtattcggtttttggggccg cgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggccacc gagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatcgcc ccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagtggccgcttcccggccctg ctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaaggaa aagggcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctcgatta gttctcaagctttggagtacgtcgtctttaggttgggggaggggttttatgcgatggagtttccccacactgagtgg gtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggatcttggtt cattctcaagcctcagacagtggttcaaagtttttcttccatttcaggtgtcgtgaccctagcgctacctctagagc caccatgaccggtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggccgtacgcaccctc gccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatccggaccgccacatcgagcgggtcacc gagctgcaagaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacgacggcgcc Guide_ gccgtggcgtctggaccacgccggagagcgtcgaagcgggggcggtgttcgccgagatcggcccgcgcatg #040 gccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcctcctggcgccgcaccggcccaa ggagcccgcgtggttcctggccaccgtcggagtctcgcccgaccaccagggcaagggtctgggcaggccgtc gtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgcaa cctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctgg tgcatgacccgcaagcccggtgcctgaacgcgttaagtcgacaatcaactctggattacaaaatttgtgaaagatt gactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcc cgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaa cgtggcgtggtgtgcactgtgttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttc cgggacttcgctttccccctccctattgccacggcggaactcatcgccgcctgcctgcccgctgctggacagg ggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtg tgccacctggattcgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgc ggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccg cctccccgcgtcgactttaagaccaatgacttacaaggcagctgtagatcttagccacttttaaaagaaaagggg gactggaagggctaattcactcccaacgaagacaagatttcaccattatcgtttcagacccacctcccaaccccg aggggacccagagagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataat tagaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtataatttcttgggtagtttg cagtttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtattcgatttcttggctttatatatct tgtggaaaggacgaggtaccgtaatttctactcttgtagatgctcacctattagcggctaaggataccaaactgtcc ccagaacagtaatttctactgtcgtagatgctttaaggccggtctagcaatcccaatctgtgtagttagcagctttttt gaattcaagcttggcgtaactagatcttgagacactgctttttgcttgtactgggtctctctggttagaccagatctga gcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgctcaagtag tgtgtgcccgtctgttgtgtgactctggtactagagatccctcagacccttttagtcagtgtggaaaatctctagcag tacgtatagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaac ttgtttattgcagcttataatggtacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcat

[0844]

[0845] tctagtgtggttgtccaaactcatcaatgtatctatcatgtctggctctagctatcccgcccctaactccgcccatc ccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgag gccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctagggacgtacccaattcgc ctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacc caacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgccc ttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcgcgggtgtgg tggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcg ccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgattagtgctttacggcac ctcgaccccaaaaaacttgatagggtgatggttcacgtagtgggcatcgccctgatagacggtttttcgccctttg acgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttt tgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaatttta acaaaatattaacgcttacaatttagtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaat acattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatga gtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgttttgctcacccagaaacgctggt gaaagtaaaaatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatc cttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatccc gtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccgtc acagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacact gcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcat gtaactcgcctgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtacaccacgatgcct gtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatag actggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataa atctggagccggtgagcgtgggtctcgcggtatcattgcagcacggggccagatggtaagccctcccgtatcgt agttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcac Guide_ tgattaagcatggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaag #040 gatctaggtgaagatcctttttgtaatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagacc ccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccac cgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagc gcagataccaaatatgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacata cctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtctaccgggttggactcaaga cgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcga acgactacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaagg cggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgc ctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctgtcaggggggcgg agcctatggaaaaacgccagcaacgcggcctttacggttcctggccttttgctggccttttgctcacatgttctttc ctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaac gaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgccaatacgcaaaccgcctctccccgcgc gttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaatt aatgtgagttagctcactcattaggcaccccaggcttacactttatgcttccggctcgtatgttgtgtggaattgtga gcggataacaattcacacaggaaacagtatgaccatgattacgccaagcgcgcaattaaccctcactaaaggg aacaaaagctggagctgcaagcttaatgtagtcttatgcaatactcttgtagtcttgcaacatggtaacgatgagtta gcaacatgccttacaaggagagaaaaagcaccgtgcatgccgattggtggaagtaaggtggtacgatcgtgcct tattaggaaggcaacgacgggtctgacatggattggacgaaccactgaattgccgcattgcagagatattgtattt aagtgcctagctcgatacataaacgggtctctctggttagaccagatctgagcctgggagctctctggctaactag ggaacccactgctaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgacttgg taactagagatccctcagaccctttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaa gcgaaagggaaaccagaggagctctctcgacgcaggactcggctgctgaagcgcgcacggcaagaggcga ggggcggcgactggtgagtacgccaaaaatttgactagcggaggctagaaggagagagatggggcgagag cgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaa

[0846]

[0847] tataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacat cagaaggctgtagacaaatactgggacagctacaaccatcccttcgacaggatcagaagaacttagatcattata taatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagat agaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggaggagga gatatgagggacaattggagaagtgaattatataatataaagtagtaaaaattgaaccattaggagtagcacccac caaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttg Guide ggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattatgtctggta #040 tagtgcagcagcagaacatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcat caagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggtt gctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaacagatttgga tcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatcg caaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaaca taacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggttaagaatagttttgctgtac tttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgagg

[0848]

[0849] ggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagt gaacggatctcgacggtatcggttaacttttaaaagaaaaggggggattggggggtcagtgcaggggaaagaa tagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttatcgat gagtaattcatacaaaaggactcgcccctgccttggggaatcccagggaccgtcgttaaactcccactaacgtag aacccagagatcgctgcgttcccgccccctcacccgccgctctcgtcatcactgaggtggagaagagcatgcgt gaggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtc ggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcc tttttcccgagggtgggggagaaccgatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgcc gccagaacacaggtaagtgccgtgtgtggtcccgcgggcctggcctctttacgggtatggcccttgcgtgcct tgaattacttccacgcccctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagt tcgaggcttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcttgggcgctggggccgccgc gtgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctg ctgcgacgctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtattcggttttggggc cgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggcc accgagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatc gccccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagtggccgcttcccggc cctgctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaa ggaaaagggcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctc gattagttctcaagctttggagtacgtcgtctttaggttgggggaggggttttatgcgatggagtttccccacactg agtgggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggatc ttggttcattctcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtcgtgaccctagcgctacctcta gagccaccatgaccggtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggccgtacgca ccctcgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatccggaccgccacatcgagcggg tcaccgagctgcaagaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacgacgg Guide_ cgccgccgtggcgtctggaccacgccggagagcgtcgaagcgggggcggtgttcgccgagatcggcccgcg #041 catggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcctcctggcgccgcaccggc ccaaggagcccgcgtggttcctggccaccgtcggagtctcgcccgaccaccagggcaagggtctgggcagg ccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccc cgcaacctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgca cctggtgcatgacccgcaagcccggtgcctgaacgcgttaagtcgacaatcaactctggattacaaaatttgtgaa agattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattg cttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcag gcaacgtggcgtggtgtgcactgtgttgctgacgcaacccccactggttggggcattgccaccacctgtcagctc ctttccgggactttcgctttccccctccctatgccacggcggaactcatcgccgcctgccttgcccgctgctggac aggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcct gtgttgccacctggattcgcgcgggacgtcctctgctacgtcccttcggccctcaatccagcggaccttccttccc gcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggc cgcctccccgcgtcgactttaagaccaatgacttacaaggcagctgtagatcttagccacttttaaaagaaaagg gggactggaagggctaattcactcccaacgaagacaagatttcaccattatcgtttcagacccacctcccaaccc cgaggggacccagagagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagat aattagaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtataatttcttgggtagt ttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggcttatata tcttgtggaaaggacgaggtaccgtaatttctactcgctttaaggccggtcctagcaatgtagatataccaaactgt ccccagaacagtaatttctactggctcacctattagcggctaaggtgtagattcccaatctgtgtagttagcagctttt ttgaattcaagcttggcgtaactagatcttgagacactgctttttgcttgtactgggtctctctggttagaccagatctg agcctgggagctctctggctaactagggaacccactgctaagcctcaataaagctgccttgagtgcttcaagta gtgtgtgcccgtctgttgtgtgactctggtactagagatccctcagacccttttagtcagtgtggaaaatctctagca gtacgtatagtagttcatgtcatcttattattcagtattataacttgcaaagaaatgaatatcagagagtgagaggaa cttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgca

[0850]

[0851] ttctagttgtggttgtccaaactcatcaatgtatcttatcatgtctggctctagctatcccgcccctaactccgcccatc ccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgag gccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctagggacgtacccaattcgc ctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacc caacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgccc ttcccaacagttgcgcagcctgaatggcg...

Claims

What is claimed is:CLAIMS1. A method for multiplexed assessment of perturbations with single cell molecular resolution, the method comprising:(a) delivering a vector encoding a pre-crRNA array comprising a barcode and multiple crRNAs to an initial population of cells, wherein cells of the initial population comprise a repressible Cast 2 enzyme;(b) culturing the initial population of cells to effect Casl2 enzyme-mediated perturbation, thereby producing a population of perturbed cells;(c) repressing pre-crRNA processing activity of the repressible Casl2 enzyme; and (d) analyzing RNA sequences of individual perturbed cells of the population of perturbed cells using a single-cell gene expression technique.

2. The method of claim 1, wherein individual cells of the initial population of cells receive at least one perturbation, and the barcode of the pre-crRNA array comprises a sequence identifying the perturbation.

3. The method of claim 1 or 2, wherein the perturbations are selected from genetic perturbations and epigenetic perturbations.

4. The method of claim 1, wherein the genetic perturbations and epigenetic perturbations are selected from deletions, insertions, and substitutions.

5. The method of any preceding claim, wherein the multiple crRNAs target a single genomic locus of interest, optionally a gene of interest.

6. Tire method of any preceding claim, wherein the multiple crRNAs target at least two different genomic loci of interest, optionally two or more genes of interest.

7. The method of any of claims 1-4, wherein the multiple crRNAs target two or more loci within the same genes of interest.

8. lire method of any preceding claim, wherein each crRNA of the pre-crRNA array comprises, in the following order, a direct repeat (DR) domain, a Spacer domain, and a 3' end.

9. The method of claim 7, w'herein each crRNA of the pre-crRNA array further comprises a capture sequence.

10. The method of claim 8, wherein the capture sequence is at the 3' end of each crRNA.

11. The method of any preceding claim, wherein (a) comprises delivering a pooled library of vectors, each encoding a pre-crRNA array comprising multiple crRNAs to the initial population of cells.

12. The method of claim 11, wherein the repressible Casl 2 enzyme comprises a degron domain, optionally, wherein the degron domain is a C-tenninal FKBP12E,6Vfusion domain, and (c) comprises culturing tlie initial population of cells in tlie presence of a dTag small molecule dTagVl.

13. The method of any preceding claim, wherein the repressible Casl 2 enzyme is selected from a repressible Casl2a, Casl2b, Casl2c, Casl2d, Casl2e, Casl2f, or Casl2g enzyme.

14. The method of claim 13, wherein the repressible Casl2 enzyme is a repressible Casl2a enzyme, optionally a repressible enhanced Acidaminococcus sp. Casl2a (enAs)Cas!2a enzyme, and further optionally wherein the repressible (enAs)Cas!2a enzyme comprises the amino acid sequence of SEQ ID NO: 1.

15. lire method of any preceding claim, wherein the repressible Casl2a enzyme is a repressible deactivated Casl2a (dCas!2a) enzyme.

16. The method of claim 15, wherein the repressible dCas!2a enzyme is linked to a transcription repressor domain, optionally selected from KRAB (Kriippel -associated box), SID (Sin3 Interaction Domain), MeCP2 (Methyl-CpG-Binding Protein 2), LSD1 (Lysine-Specific Demethylase 1), dCas-Mxil, CoREST, Ezh2 (Enhancer of Zeste Homolog 2), HP1 (Heterochromatin Protein 1), REST (Repressor Element-1 Silencing Transcription Factor), Polycomb Repressor Complex (PRC2), ZIM3 (Zinc Finger Imprinted 3), DNMT3A (DNA methyltransferase 3 alpha), and DNMT3L (DNA methyltransferase 3 like).

17. The method of claim 15, wherein the repressible dCas!2a enzyme is linked to a transcription activation domain, optionally selected from VP64, p65, Rta (Replication and Transcription Activator), VPR (VP64-p65-Rta), SunTag, HSF1 (Heat Shock Factor 1), P300 Core, SAM (Synergistic Activation Mediator), and ET1 (Epstein-Barr Virus EBNA-LP Transactivation Domain),18. The method of any preceding claim, wherein the vector is a non- viral vector.

1. The method of claim 18, wdierem the non-viral vector is selected from plasmid DNA, minicircle DNA, transposons, and artificial chromosomes.

20. Tire method of any of claims 1-17, wherein the vector is a viral vector,21. The method of claim 20, wherein the viral vector is selected from lentiviral vectors, adenoviral vectors, adeno-associated viral (AAV) vectors, retroviral vectors, herpes simplex virus (HSV) vectors, sendai virus (SeV) vectors, and baculovirus vectors.

22. The method of any of claims 18-21 wherein delivering of the vector comprises transfecting or transducing cells of the population with the vector such that single cells of the initial population receive a single pre-crR A array.

23. The method of any preceding claim, wherein the single-cell gene expression technique comprises single-cell RNA sequencing.

24. The method of any preceding claim further comprising determining measured differences in the RNA sequences that are correlated to perturbations detected for individual perturbed cells.

25. A m ethod for multiplexed assessment of perturbations with single cell molecular resolution, the method comprising:(a) culturing an initial population of cells comprising (i) a pre-crRNA array comprising a barcode and multiple crRNAs and (ii) a repressible Casl 2a enzyme to effect Cas 12a enzyme-mediated perturbation, thereby producing a population of perturbed cells; and (b) repressing pre-crRNA processing activity of the repressi ble Cas 12a enzyrn e 26. The method of claim 25 further comprising analyzing RNA sequences of individual perturbed cells of the population of perturbed cells using a single-cell gene expression technique.

27. A Cas 12a enzyme linked to a repressible domain.

28. The Cas 12a enzyme of claim 27, wherein the repressible domain comprises a degron domain.

29. lire Casl2a enzyme of claim 28 comprising an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO:

130. The Casl2a enzyme of claim 29 comprising an amino acid sequence having a at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 1.

31. A composition comprising a vector encoding a repressible Cas 12a enzyme and a vector encoding a pre-crRNA array.

32. The composition of claim 31 comprising multiple vectors, each encoding a pre-crRNA array.

33. A cell comprising a vector encoding a pre-crRNA array, wherein the cell expresses a repressible Cas 12a enzyme.

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