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Definitions

• a eukaryotic cell is transfected with a two component system including RNA complementary to genomic DNA and an enzyme that interacts with the RNA.
• the RNA and the enzyme are expressed by the cell.
• the RNA of the RNA/enzyme complex then binds to complementary genomic DNA.
• the enzyme then performs a function, such as cleavage of the genomic DNA.
• the RNA includes between about 10 nucleotides to about 250 nucleotides.
• the RNA includes between about 20 nucleotides to about 100 nucleotides.
• the enzyme may perform any desired function in a site specific manner for which the enzyme has been engineered.
• the eukaryotic cell is a yeast cell, plant cell or mammalian cell.
• the enzyme cleaves genomic sequences targeted by RNA sequences (see references (4-6)), thereby creating a genomically altered eukaryotic cell.
• the present disclosure provides a method of genetically altering a human cell by including a nucleic acid encoding an RNA complementary to genomic DNA into the genome of the cell and a nucleic acid encoding an enzyme that performs a desired function on
• the RNA and the enzyme are expressed, According to one aspect, the RNA hybridizes with complementary genomic DNA. According to one aspect, the enzyme is activated to perform a desired function, such as cleavage, in a site specific manner when the RNA is hybridized to the complementary genomic DNA. According to one aspect, the RNA and the enzyme are components of a bacterial Type II CRISPR system.
• a method of altering a eukaryotic cell including transfecting the eukaryotic cell with a nucleic acid encoding RNA complementary to genomic DNA of the eukaryotic cell, transfecting the eukaryotic cell with a nucleic acid encoding an enzyme that interacts with the RNA and cleaves the genomic DNA in a site specific manner, wherein the cell expresses the RNA and the enzyme, the RNA binds to complementary genomic DNA and the enzyme cleaves the genomic DNA in a site specific manner.
• the enzyme is Cas9 or modified Cas9 or a homolog of Cas9.
• the eukaryotic cell is a yeast cell, a plant cell or a mammalian cell.
• the RNA includes between about 10 to about 250 nucleotides.
• the RNA includes between about 20 to about 100 nucleotides.
• a method of altering a human cell including transfecting the human cell with a nucleic acid encoding RNA complementary to genomic DNA of the eukaryotic cell, transfecting the human cell with a nucleic acid encoding an enzyme that interacts with the RNA and cleaves the genomic DNA in a site specific manner, wherein the human cell expresses the RNA and the enzyme, the RNA binds to complementary genomic DNA and the enzyme cleaves the genomic DNA in a site specific manner.
• the enzyme is Cas9 or modified Cas9 or a homolog of Cas9.
• the RNA includes between about 10 to about 250 nucleotides.
• the RNA includes between about 20 to about 100 nucleotides.
• a method of altering a eukaryotic cell at a plurality of genomic DNA sites including transfecting the eukaryotic cell with a plurality of nucleic acids encoding RNAs complementary to different sites on genomic DNA of the eukaryotic cell, transfecting the eukaryotic cell with a nucleic acid encoding an enzyme that interacts with the RNA and cleaves the genomic DNA in a site specific manner, wherein the cell expresses the RNAs and the enzyme, the RNAs bind to complementary genomic DNA and the enzyme cleaves the genomic DNA in a site specific manner.
• the enzyme is Cas9.
• the eukaryotic cell is a yeast cell, a plant cell or a mammalian cell.
• the RNA includes between about 10 to about 250 nucleotides.
• the RNA includes between about 20 to about 100 nucleotides.
• a human codon-optimized version of the Cas9 protein bearing a C-terminus SV40 nuclear localization signal is synthetized and cloned into a mammalian expression system (Fig. 1A and Fig. 3 A).
• Figure 1 is directed to genome editing in human cells using an engineered type II CRISPR system.
• RNA-guided gene targeting in human cells involves co-expression of the Cas9 protein bearing a C-terminus SV40 nuclear localization signal with one or more guide RNAs (gRNAs) expressed from the human U6 polymerase III promoter.
• gRNAs guide RNAs
• Cas9 unwinds the DNA duplex and cleaves both strands upon recognition of a target sequence by the gRNA, but only if the correct protospacer-adjacent motif (PAM) is present at the 3' end.
• Any genomic sequence of the form GN 2 oGG can in principle be targeted.
• a genomically integrated GFP coding sequence is disrupted by the insertion of a stop codon and a 68bp genomic fragment from the AAVS l locus.
• Restoration of the GFP sequence by homologous recombination (HR) with an appropriate donor sequence results in GFP + cells that can be quantitated by FACS.
• Tl and T2 gRNAs target sequences within the AAVS l fragment.
• Binding sites for the two halves of the TAL effector nuclease heterodimer are underlined.
• crRNA-tracrRNA fusion transcripts are expressed, hereafter referred to as guide RNAs (gRNAs), from the human U6 polymerase III promoter.
• gRNAs are directly transcribed by the cell. This aspect advantageously avoids reconstituting the RNA processing machinery employed by bacterial CRISPR systems (Fig. 1A and Fig. 3B) (see references (4, 7-9)).
• a method is provided for altering genomic DNA using a U6 transcription initiating with G and a PAM (protospacer-adjacent motif) sequence -NGG following the 20 bp crRNA target.
• the target genomic site is in the form of GN 2 oGG (See Fig. 3C).
• a GFP reporter assay (Fig. IB) in 293T cells was developed similar to one previously described (see reference (10)) to test the functionality of the genome engineering methods described herein.
• a stable cell line was established bearing a genomically integrated GFP coding sequence disrupted by the insertion of a stop codon and a 68bp genomic fragment from the AAVS l locus that renders the expressed protein fragment non-fluorescent.
• Homologous recombination (HR) using an appropriate repair donor can restore the normal GFP sequence, which allows one to quantify the resulting GFP + cells by flow activated cell sorting (FACS).
• FACS flow activated cell sorting
• a method is provided of homologous recombination (HR).
• RNA-mediated editing process was notably rapid, with the first detectable GFP + cells appearing -20 hours post transfection compared to -40 hours for the AAVS l TALENs.
• a native locus was modified.
• gRNAs were used to target the AAVS l locus located in the PPP1R12C gene on chromosome 19, which is ubiquitously expressed across most tissues (Fig. 2A) in 293Ts, K562s, and PGP1 human iPS cells (see reference (12)) and analyzed the results by next-generation sequencing of the targeted locus.
• Figure 2 is directed to RNA-guided genome editing of the native AAVS l locus in multiple cell types.
• Tl (red) and T2 (green) gRNAs target sequences in an intron of the PPP1R12C gene within the chromosome 19 AAVS l locus.
• PCR assay three days post transfection demonstrates that only cells expressing the donor, Cas9 and T2 gRNA exhibit successful HR events.
• successful HR was confirmed by Sanger sequencing of the PCR amplicon showing that the expected DNA bases at both the genome-donor and donor-insert boundaries are present.
• Figure 2F successfully targeted clones of 293T cells were selected with puromycin for 2 weeks. Microscope images of two representative GFP+ clones is shown (scale bar is 100 microns).
• HR is used to integrate either a dsDNA donor construct (see reference (13)) or an oligo donor into the native AAVS l locus (Fig. 2C, Fig. 12).
• HR-mediated integration was confirmed using both approaches by PCR (Fig. 2D, Fig. 12) and Sanger sequencing (Fig. 2E).
• 293T or iPS clones were readily derived from the pool of modified cells using puromycin selection over two weeks (Fig. 2F, Fig. 12).
• an RNA-guided genome editing system which can readily be adapted to modify other genomic sites by simply modifying the sequence of the gRNA expression vector to match a compatible sequence in the locus of interest.
• 190,000 specifically gRNA-targetable sequences targeting about 40.5% exons of genes in the human genome were generated. These target sequences were incorporated into a 200bp format compatible with multiplex synthesis on DNA arrays (see reference (14)) (Fig. 13).
• a ready genome -wide reference of potential target sites in the human genome and a methodology for multiplex gRNA synthesis is provided.
• methods are provided for multiplexing genomic alterations in a cell by using one or more or a plurality of RNA/enzyme systems described herein to alter the genome of a cell at a plurality of locations.
• target sites perfectly match the PAM sequence NGG and the 8- 12 base "seed sequence" at the 3 ' end of the gRNA.
• perfect match is not required of the remaining 8- 12 bases.
• Cas9 will function with single mismatches at the 5' end.
• the target locus 's underlying chromatin structure and epigenetic state may affect efficiency of Cas9 function.
• Cas9 homologs having higher specificity are included as useful enzymes.
• CRISPR-targetable sequences include those having different PAM requirements (see reference (9)), or directed evolution.
• inactivating one of the Cas9 nuclease domains increases the ratio of HR to NHEJ and may reduce toxicity (Fig. 3 A, Fig. 5) (4, 5), while inactivating both domains may enable Cas9 to function as a retargetable DNA binding protein.
• Embodiments of the present disclosure have broad utility in synthetic biology (see references (21, 22)), the direct and multiplexed perturbation of gene networks (see references (13, 23)), and targeted ex vivo (see references (24-26)) and in vivo gene therapy (see reference (27)).
• a "re-engineerable organism” is provided as a model system for biological discovery and in vivo screening.
• a "re-engineerable mouse” bearing an inducible Cas9 transgene is provided, and localized delivery (using adeno- associated viruses, for example) of libraries of gRNAs targeting multiple genes or regulatory elements allow one to screen for mutations that result in the onset of tumors in the target tissue type.
• Use of Cas9 homologs or nuclease-null variants bearing effector domains (such as activators) allow one to multiplex activate or repress genes in vivo.
• DNA-arrays enables multiplex synthesis of defined gRNA libraries (refer Fig. 13); and (b) gRNAs being small in size (refer Fig. 3b) are packaged and delivered using a multitude of non- viral or viral delivery methods.
• the lower toxicities observed with "nickases” for genome engineering applications is achieved by inactivating one of the Cas9 nuclease domains, either the nicking of the DNA strand base-paired with the RNA or nicking its complement. Inactivating both domains allows Cas9 to function as a retargetable DNA binding protein.
• the Cas9 retargetable DNA binding protein is attached
• nuclease domains such as Fokl to enable 'highly specific' genome editing contingent upon dimerization of adjacent gRNA-Cas9 complexes
• the transcriptional activation and repression components can employ CRISPR systems naturally or synthetically orthogonal, such that the gRNAs only bind to the activator or repressor class of Cas. This allows a large set of gRNAs to tune multiple targets.
• the use of gRNAs provide the ability to multiplex than mRNAs in part due to the smaller size - 100 vs. 2000 nucleotide lengths respectively. This is particularly valuable when nucleic acid delivery is size limited, as in viral packaging. This enables multiple instances of cleavage, nicking, activation, or repression - or combinations thereof.
• the ability to easily target multiple regulatory targets allows the coarse-or- fine-tuning or regulatory networks without being constrained to the natural regulatory circuits downstream of specific regulatory factors (e.g. the 4 mRNAs used in reprogramming fibroblasts into IPSCs). Examples of multiplexing applications include:
• Multiplex cis-regulatory element (CRE signals for transcription, splicing, translation, RNA and protein folding, degradation, etc.) mutations in a single cell (or a collection of cells) can be used for efficiently studying the complex sets of regulatory interaction that can occur in normal development or pathological, synthetic or pharmaceutical scenarios.
• the CREs are (or can be made) somewhat orthogonal (i.e. low cross talk) so that many can be tested in one setting— e.g. in an expensive animal embryo time series.
• One exemplary application is with RNA fluorescent in situ sequencing (FISSeq).
• CRE mutations and/or epigenetic activation or repression of CREs can be used to alter or reprogram iPSCs or ESCs or other stem cells or non-stem cells to any cell type or combination of cell types for use in organs-on-chips or other cell and organ cultures for purposes of testing pharmaceuticals (small molecules, proteins, RNAs, cells, animal, plant or microbial cells, aerosols and other delivery methods), transplantation strategies, personalization strategies, etc.
• Repetitive elements or endogenous viral elements can be targeted with engineered Cas + gRNA systems in microbes, plants, animals, or human cells to reduce deleterious transposition or to aid in sequencing or other analytic genomic/transcriptomic/proteomic/diagnostic tools (in which nearly identical copies can be problematic).
• embodiments of the present disclosure utilize short RNA to identify foreign nucleic acids for activity by a nuclease in a eukaryotic cell.
• a eukaryotic cell is altered to include within its genome nucleic acids encoding one or more short RNA and one or more nucleases which are activated by the binding of a short RNA to a target DNA sequence.
• exemplary short RNA / enzyme systems may be identified within bacteria or archaea, such as (CRISPR)/CRISPR- associated (Cas) systems that use short RNA to direct degradation of foreign nucleic acids.
• CRISPR (“clustered regularly interspaced short palindromic repeats") defense involves acquisition and integration of new targeting "spacers” from invading virus or plasmid DNA into the CRISPR locus, expression and processing of short guiding CRISPR RNAs (crRNAs) consisting of spacer- repeat units, and cleavage of nucleic acids (most commonly DNA) complementary to the spacer.
• spacers new targeting "spacers” from invading virus or plasmid DNA into the CRISPR locus
• crRNAs short guiding CRISPR RNAs
• nucleic acids most commonly DNA
• Type II Three classes of CRISPR systems are generally known and are referred to as Type I, Type II or Type III).
• a particular useful enzyme according to the present disclosure to cleave dsDNA is the single effector enzyme, Cas9, common to Type II. (See reference (7)).
• the Type II effector system consists of a long pre-crRNA transcribed from the spacer-containing CRISPR locus, the multifunctional Cas9 protein, and a tracrRNA important for gRNA processing.
• the tracrRNAs hybridize to the repeat regions separating the spacers of the pre-crRNA, initiating dsRNA cleavage by endogenous RNase III, which is followed by a second cleavage event within each spacer by Cas9, producing mature crRNAs that remain associated with the tracrRNA and Cas9.
• eukaryotic cells of the present disclosure are engineered to avoid use of RNase III and the crRNA processing in general. See reference (2).
• the enzyme of the present disclosure such as Cas9 unwinds the DNA duplex and searches for sequences matching the crRNA to cleave.
• Target recognition occurs upon detection of complementarity between a "protospacer” sequence in the target DNA and the remaining spacer sequence in the crRNA.
• Cas9 cuts the DNA only if a correct protospacer-adjacent motif (PAM) is also present at the 3' end.
• PAM protospacer-adjacent motif
• different protospacer-adjacent motif can be utilized.
• the S. pyogenes system requires an NGG sequence, where N can be any nucleotide.
• S. thermophilus Type II systems require NGGNG (see reference (3)) and NNAGAAW (see reference (4)), respectively, while different S. mutans systems tolerate NGG or NAAR (see reference (5)).
• DSB formation also occurs towards the 3 ' end of the protospacer. If one of the two nuclease domains is inactivated, Cas9 will function as a nickase in vitro (see reference (2)) and in human cells (see Figure 5).
• the specificity of gRNA-directed Cas9 cleavage is used as a mechanism for genome engineering in a eukaryotic cell.
• hybridization of the gRNA need not be 100 percent in order for the enzyme to recognize the gRNA/DNA hybrid and affect cleavage.
• Some off-target activity could occur.
• the S. pyogenes system tolerates mismatches in the first 6 bases out of the 20bp mature spacer sequence in vitro.
• greater stringency may be beneficial in vivo when potential off-target sites matching (last 14 bp) NGG exist within the human reference genome for the gRNAs.
• the effect of mismatches and enzyme activity in general are described in references (9), (2), (10), and (4).
• specificity may be improved.
• AT-rich target sequences may have fewer off- target sites. Carefully choosing target sites to avoid pseudo-sites with at least 14bp matching sequences elsewhere in the genome may improve specificity.
• the use of a Cas9 variant requiring a longer PAM sequence may reduce the frequency of off-target sites.
• Directed evolution may improve Cas9 specificity to a level sufficient to completely preclude off-target activity, ideally requiring a perfect 20bp gRNA match with a minimal PAM. Accordingly, modification to the Cas9 protein is a representative embodiment of the present disclosure. As such, novel methods permitting many rounds of evolution in a short timeframe (see reference (11) and envisioned. CRISPR systems useful in the present disclosure are described in references (12, 13).
• the Cas9 gene sequence was human codon optimized and assembled by hierarchical fusion PCR assembly of 9 500bp gBlocks ordered from IDT.
• Fig. 3A for the engineered type II CRISPR system for human cells shows the expression format and full sequence of the cas9 gene insert.
• the RuvC-like and HNH motifs, and the C-terminus SV40 NLS are respectively highlighted by blue, brown and orange colors.
• Cas9_D 10A was similarly constructed. The resulting full-length products were cloned into the pcDNA3.3-TOPO vector (Invitrogen).
• Fig. 3B shows the U6 promoter based expression scheme for the guide RNAs and predicted RNA transcript secondary structure. The use of the U6 promoter constrains the 1 st position in the RNA transcript to be a 'G' and thus all genomic sites of the form GN 2 oGG can be targeted using this approach.
• Fig. 3C shows the 7 gRNAs used.
• the vectors for the HR reporter assay involving a broken GFP were constructed by fusion PCR assembly of the GFP sequence bearing the stop codon and 68bp AAVS l fragment (or mutants thereof; see Fig. 6), or 58bp fragments from the DNMT3a and DNMT3b genomic loci (see Fig. 8) assembled into the EGIP lentivector from Addgene (plasmid #26777). These lentivectors were then used to establish the GFP reporter stable lines.
• TALENs used in this study were constructed using the protocols described in (14). All DNA reagents developed in this study are available at Addgene.
• HEK 293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) high glucose supplemented with 10% fetal bovine serum (FBS, Invitrogen), penicillin/streptomycin (pen/strep, Invitrogen), and non-essential amino acids (NEAA, Invitrogen). All cells were maintained at 37°C and 5% CO 2 in a humidified incubator.
• DMEM Dulbecco's modified Eagle's medium
• FBS fetal bovine serum
• pen/streptomycin penicillin/streptomycin
• NEAA non-essential amino acids
• PGP1 iPS cells were cultured in Rho kinase (ROCK) inhibitor (Calbiochem) 2h before nucleofection.
• ROCK Rho kinase
• Cells were harvest using TrypLE Express (Invitrogen) and 2* 10 6 cells were resuspended in P3 reagent (Lonza) with ⁇ g Cas9 plasmid, ⁇ g gRNA and/or ⁇ g DNA donor plasmid, and nucleofected according to manufacturer's instruction (Lonza).
• Cells were subsequently plated on an mTeSRl -coated plate in mTeSRl medium supplemented with ROCK inhibitor for the first 24h.
• the targeting efficiency was assessed as follows. Cells were harvested 3 days after nucleofection and the genomic DNA of ⁇ 1 X 10 6 cells was extracted using prepGEM (ZyGEM). PCR was conducted to amplify the targeting region with genomic DNA derived from the cells and amplicons were deep sequenced by MiSeq Personal Sequencer (Illumina) with coverage >200,000 reads. The sequencing data was analyzed to estimate NHEJ efficiencies.
• the reference AAVS 1 sequence analyzed is:
• the PCR primers for amplifying the targeting regions in the human genome are:
• a set of gRNA gene sequences that maximally target specific locations in human exons but minimally target other locations in the genome were determined as follows. According to one aspect, maximally efficient targeting by a gRNA is achieved by 23nt sequences, the 5 '-most 20nt of which exactly complement a desired location, while the three 3 '-most bases must be of the form NGG. Additionally, the 5 '-most nt must be a G to establish a pol-III transcription start site.
• mispairing of the six 5'-most nt of a 20bp gRNA against its genomic target does not abrogate Cas9-mediated cleavage so long as the last 14nt pairs properly, but mispairing of the eight 5 '-most nt along with pairing of the last 12 nt does, while the case of the seven 5-most nt mispairs and 13 3' pairs was not tested.
• one condition was that the case of the seven 5 '-most mispairs is, like the case of six, permissive of cleavage, so that pairing of the 3 '-most 13nt is sufficient for cleavage.
• Coding exon locations in this BED file comprised a set of 346089 mappings of RefSeq mRNA accessions to the hgl9 genome. However, some RefSeq mRNA accessions mapped to multiple genomic locations (probable gene duplications), and many accessions mapped to subsets of the same set of exon locations (multiple isoforms of the same genes).
• RefSeq mRNA mappings were clustered so that any two RefSeq accessions (including the gene duplicates distinguished in (ii)) that overlap a merged exon region are counted as a single gene cluster, the 189864 exonic specific CRISPR sites target 17104 out of 18872 gene clusters (-90.6% of all gene clusters) at a multiplicity of ⁇ 1 1.1 per targeted gene cluster.
• the database can be refined by correlating performance with factors, such as base composition and secondary structure of both gRNAs and genomic targets (20, 21), and the epigenetic state of these targets in human cell lines for which this information is available (22).
• factors such as base composition and secondary structure of both gRNAs and genomic targets (20, 21), and the epigenetic state of these targets in human cell lines for which this information is available (22).
• the target sequences were incorporated into a 200bp format that is compatible for multiplex synthesis on DNA arrays (23, 24).
• the method allows for targeted retrieval of a specific or pools of gRNA sequences from the DNA array based oligonucleotide pool and its rapid cloning into a common expression vector (Fig. 13 A). Specifically, a 12k oligonucleotide pool from CustomArray Inc. was synthesized. Furthermore, gRNAs of choice from this library (Fig. 13B) were successfully retrieved. We observed an error rate of -4 mutations per 1 OOObp of synthesized DNA.
• RNA-guided genome editing requires both Cas9 and guide RNA for successful targeting
• Each of the lines was then targeted by one of the following 4 reagents: a GFP-ZFN pair that can target all cell types since its targeted sequence was in the flanking GFP fragments and hence present in along cell lines; a AAVS 1 TALEN that could potentially target only the wt-AAVS 1 fragment since the mutations in the other two lines should render the left TALEN unable to bind their sites; the Tl gRNA which can also potentially target only the wt-AAVS 1 fragment, since its target site is also disrupted in the two mutant lines; and finally the T2 gRNA which should be able to target all 3 cell lines since, unlike the Tl gRNA, its target site is unaltered among the 3 lines.
• RNA-guided genome editing is target sequence specific, and demonstrates similar targeting efficiencies as ZFNs or TALENs Similar to the GFP reporter assay described in Fig. IB, two 293T stable lines each bearing a distinct GFP reporter construct were developed. These are distinguished by the sequence of the fragment insert (as indicated in Figure 8). One line harbored a 58bp fragment from the DNMT3a gene while the other line bore a homologous 58bp fragment from the DNMT3b gene. The sequence differences are highlighted in red.
• PGP1 Human iPS cells
• DPBs double-strand breaks
• Panel 1 Deletion rate detected at targeting region. Red dash lines: boundary of Tl RNA targeting site; green dash lines: boundary of T2 RNA targeting site. The deletion incidence at each nucleotide position was plotted in black lines and the deletion rate as the percentage of reads carrying deletions was calculated.
• Panel 2 Insertion rate detected at targeting region. Red dash lines: boundary of Tl RNA targeting site; green dash lines: boundary of T2 RNA targeting site.
• K562 cells were nucleated with constructs indicated in the left panel of Figure 10. 4 days after nucleofection, NHEJ rate was measured by assessing genomic deletion and insertion rate at DSBs by deep sequencing.
• Panel 1 Deletion rate detected at targeting region. Red dash lines: boundary of Tl RNA targeting site; green dash lines: boundary of T2 RNA targeting site. The deletion incidence at each nucleotide position was plotted in black lines and the deletion rate as the percentage of reads carrying deletions was calculated.
• Panel 2 Insertion rate detected at targeting region. Red dash lines: boundary of Tl RNA targeting site; green dash lines: boundary of T2 RNA targeting site.
• 293T cells were transfected with constructs indicated in the left panel of Figure 11. 4 days after nucleofection, NHEJ rate was measured by assessing genomic deletion and insertion rate at DSBs by deep sequencing.
• Panel 1 Deletion rate detected at targeting region. Red dash lines: boundary of Tl RNA targeting site; green dash lines: boundary of T2 RNA targeting site. The deletion incidence at each nucleotide position was plotted in black lines and the deletion rate as the percentage of reads carrying deletions was calculated.
• Panel 2 Insertion rate detected at targeting region. Red dash lines: boundary of Tl RNA targeting site; green dash lines: boundary of T2 RNA targeting site.
• the incidence of insertion at the genomic location where the first insertion junction was detected was plotted in black lines and the insertion rate as the percentage of reads carrying insertions was calculated.
• Panel 3 Deletion size distribution. The frequencies of different size deletions among the whole NHEJ population was plotted.
• Panel 4 insertion size distribution. The frequencies of different sizes insertions among the whole NHEJ population was plotted. 293T targeting by both gRNAs is efficient (10-24%) and sequence specific (as shown by the shift in position of the NHEJ deletion distributions).
• PCR screen (with reference to Figure 2C) confirmed that 21/24 randomly picked 293T clones were successfully targeted.
• FIG 12B similar PCR screen confirmed 3/7 randomly picked PGPl-iPS clones were also successfully targeted.
• short 90mer oligos could also effect robust targeting at the endogenous AAVS 1 locus (shown here for K562 cells).
• a resource of about 190k bioinformatically computed unique gRNA sites targeting -40.5% of all exons of genes in the human genome was generated.
• the gRNA target sites were incorporated into a 200bp format that is compatible for multiplex synthesis on DNA arrays.
• the design allows for (i) targeted retrieval of a specific or pools of gRNA targets from the DNA array oligonucleotide pool (through 3 sequential rounds of nested PCR as indicated in the figure schematic); and (ii) rapid cloning into a common expression vector which upon linearization using an Aflll site serves as a recipient for Gibson assembly mediated incorporation of the gRNA insert fragment.
• the method was used to accomplish targeted retrieval of 10 unique gRNAs from a 12k oligonucleotide pool synthesized by CustomArray Inc.
• the CRISPR-Cas system has an adaptive immune defense system in bacteria and functions to 'cleave' invading nucleic acids.
• the CRISPR-CAS system is engineered to function in human cells, and to 'cleave' genomic DNA. This is achieved by a short guide RNA directing a Cas9 protein (which has nuclease function) to a target sequence complementary to the spacer in the guide RNA.
• the ability to 'cleave' DNA enables a host of applications related to genome editing, and also targeted genome regulation.
• the Cas9 protein was mutated to make it nuclease-null by introducing mutations that are predicted to abrogate coupling to Mg2+ (known to be important for the nuclease functions of the RuvC-like and HNH-like domains): specifically, combinations of D10A, D839A, H840A and N863A mutations were introduced.
• the thus generated Cas9 nuclease-null protein (as confirmed by its ability to not cut DNA by sequencing analysis) and hereafter referred to as Cas9R-H-, was then coupled to a transcriptional activation domain, here VP64, enabling the CRISPR-cas system to function as a RNA guided transcription factor (see Figure 14).
• the Cas9R-H-+VP64 fusion enables RNA- guided transcriptional activation at the two reporters shown. Specifically, both FACS analysis and immunofluorescence imaging demonstrates that the protein enables gRNA sequence specific targeting of the corresponding reporters, and furthermore, the resulting transcription activation as assayed by expression of a dTomato fluorescent protein was at levels similar to those induced by a convention TALE-VP64 fusion protein.
• Flexibility of the gRNA scaffold sequence to designer sequence insertions was determined by systematically assaying for a range of the random sequence insertions on the 5', middle and 3' portions of the gRNA: specifically, lbp, 5bp, lObp, 20bp, and 40bp inserts were made in the gRNA sequence at the 5', middle, and 3' ends of the gRNA (the exact positions of the insertion are highlighted in 'red' in Figure 15). This gRNA was then tested for functionality by its ability to induce HR in a GFP reporter assay (as described herein). It is evident that gRNAs are flexible to sequence insertions on the 5' and 3' ends (as measured by retained HR inducing activity).
• aspects of the present disclosure are directed to tagging of small-molecule responsive RNA aptamers that may trigger onset of gRNA activity, or gRNA visualization. Additionally, aspects of the present disclosure are directed to tethering of ssDNA donors to gRNAs via hybridization, thus enabling coupling of genomic target cutting and immediate physical localization of repair template which can promote homologous recombination rates over error- prone non-homologous end-joining.
• the following references identified in the Examples section by number are hereby incorporated by reference in their entireties for all purposes.

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