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New human chromosomal safe harbor sites for genome engineering with CRISPR/Cas9, TAL effector and homing endonucleases

Safe Harbor Sites (SHS) are genomic locations where new genes or genetic elements can be introduced without disrupting the expression or regulation of adjacent genes. We have identified 35 potential new human SHS in order to substantially expand SHS options beyond the three widely used canonical human SHS, AAVS1, CCR5 and hROSA26. All 35 potential new human SHS and the three canonical sites were assessed for SHS potential using 9 different criteria weighted to emphasize safety that were broader and more genomics-based than previous efforts to assess SHS potential. We then systematically compared and rank-ordered our 35 new sites and the widely used human AAVS1, hROSA26 and CCR5 sites, then experimentally validated a subset of the highly ranked new SHS together versus the canonical AAVS1 site. These characterizations included in vitro and in vivo cleavage-sensitivity tests; the assessment of population-level sequence variants that might confound SHS targeting or use for genome engineering; homology–dependent and –independent, SHS-targeted transgene integration in different human cell lines; and comparative transgene integration efficiencies at two new SHS versus the canonical AAVS1 site. Stable expression and function of new SHS-integrated transgenes were demonstrated for transgene-encoded fluorescent proteins, selection cassettes and Cas9 variants including a transcription transactivator protein that were shown to drive large deletions in a PAX3/FOXO1 fusion oncogene and induce expression of the MYF5 gene that is normally silent in human rhabdomyosarcoma cells. We also developed a SHS genome engineering ‘toolkit’ to enable facile use of the most extensively characterized of our new human SHS located on chromosome 4p. We anticipate our newly identified human SHS, located on 16 chromosomes including both arms of the human X chromosome, will be useful in enabling a wide range of basic and more clinically-oriented human gene editing and engineering.


Introduction
Safe harbor sites (SHS) are genomic loci where genes or other genetic elements can be safely inserted and expressed. These SHS are critical for effective human disease gene therapies; for investigating gene structure, function and regulation; and for cell marking and tracking. The most widely used human SHS were identified by serendipity (e.g., the AAVS1 adeno-associated virus insertion site on chromosome 19 1,2 ); by homology with useful SHS in other species (e.g., the human homolog of the murine Rosa26 locus 3 ); and most recently by recognition of the dispensability of a subset of human genes in most or all individuals (e.g., the CCR5 chemokine receptor gene, that when deleted confers resistance to HIV infection [4][5][6].
In order to more systematically identify and expand the number of useful human SHS, we first searched for target sites in the human genome predicted to be efficiently cleaved by the canonical genome engineering homing/meganuclease mCreI [7][8][9] . We reasoned that any potential SHS identified by the presence of a high quality mCreI site would also contain one or more adjacent cleavage sites for Cas9 and TALEN genome engineering nucleases that have less stringent targeting requirements. This initial precise anchoring of potential new safe harbor sites in the human genome would, in turn, facilitate better assessment of site safety, potential functional competence and the presence of confounding sequence variations. Our aim was to identify new human SHS that could be targeted by any of these three nuclease types, in a wide range of human cells, to broadly enable a wide range of basic science as well as clinical applications.
We report here the identification of 35 potential new human SHS, located on 16 different human chromosomes and 23 chromosome arms including both arms of the human X chromosome. These 35 new SHS and the three canonical human SHS ( AAVS1 , the human ROSA26 locus and CCR5) were assessed and rank-ordered for safety and potential utility using a comprehensive scoring system that included 8 different genomic criteria in addition to uniqueness. Several high-ranking potential new SHS were experimentally validated by PCR amplification, mCreI cleavage sensitivity and DNA sequencing, together with a demonstration of efficient editing and transgene insertion mediated by Cas9, TALEN and mCreI nucleases. SHS-specific transgene insertion by both homology-mediated as well as cleavage-dependent, likely homology-independent mechanisms was demonstrated. The most extensively characterized of these new SHS, the high-ranking SHS231 located on the proximal long arm of chromosome 4, was also shown to be functionally competent for recombinase/integrasemediated editing. Selectable, scorable and fluorescent/functional protein-encoding SHS231 transgenes were shown to be stably expressed when compared with the same transgenes inserted into the canonical AAVS1 site in a number of different human cell lines. The SHS231 engineering toolkit will allow others to make rapid use of this enhanced chromosome 4 SHS for both basic and clinically-oriented genome engineering applications.

SHS identification and experimental validation
In order to identify potential new human SHS, we first searched the human genome for high quality matches to the target sequence of the canonical homing endonuclease mCreI. We reasoned that a SHS identified by a highly cleavage-sensitive mCreI target site or variant would also contain one or more adjacent cleavage sites for Cas9 and TALEN-based nucleases that have less stringent targeting requirements. The well-defined mCreI site would also anchor the search of adjacent chromosomal DNA to assess and rank-order SHS suitability based on criteria for site safety, functional competence and the presence of potentially confounding sequence variations. This search was initiated by using detailed information on the cleavage specificity of mCreI that quantified the contribution of each basepair in the mCreI target site sequence. This position weight matrix was used to construct a list of 128 target site sequence variants predicted to be cleaved with ≥90% of the efficiency of the native mCreI site [11][12][13][14][15][16] (Fig. 1A and 1B). These 128 mCreI target site variants were FASTA-formatted and uploaded to the NCBI BLAST search engine (http://blast.ncbi.nlm.nih.gov/) in order to identify target site matches in the human genome (GRCh37/hg19) using the following BLAST parameters: optimize for 'Highly similar sequences (megablast)'; max target seqs = 50; short queries: 'adjust for short sequences'; expect threshold = 1; word size = 7; match/mismatch: 4, −5; and gap cost: existence=12/extension= 8. All resulting genomic target site matches of ≥95% identity (19/20 or 20/20 bp matches versus the canonical mCreI target site) were subsequently evaluated as potential new safe harbor sites.
Potential new human SHS identified by BLAST search and the canonical human SHS AAVS1 , HsROSA26 and CCR5 were then evaluated for SHS potential by 8 criteria in addition to site uniqueness that assessed site safety, accessibility and functional criteria ( Fig. 1C; Table 1 and 2).
These criteria were based on several less extensive lists of criteria (e.g., proximity to known genes or regulatory elements, see, e.g. , Sadelain et al 2012 17 ), and made use of contemporary genomic data, e.g. , ENCODE Consortium project results 18 . All SHS candidates including the three canonical human SHS were evaluated as follows: sites were first searched 300 kb up-and downstream in the UCSC Genome Browser in order to identify genes or RNAs, especially any already related to cancer; proximity to any transcriptionally active region regardless of annotation; the presence of replication origins or ultra-conserved elements; location in open chromatin as assessed by nuclease sensitivity; and whether the SHS was located in a region of copy number variation 19,20 (CNV; http://genome.ucsc.edu/ ). We next used 1000 Genomes Project (1KGP) data ( http://www.ncbi.nlm.nih.gov/variation/tools/1000genomes/ ) to identify basepair-level population genetic variation within all of the mCreI-anchored SHS sites 21 (Table S2). This approach was used to provide an estimate of the fraction of SHS that would be directly accessible in individuals by mCreI (and, by extension, other genome engineering nucleases). New SHS that differed from the canonical mCreI site at 1 or more basepair positions were further assessed using the mCreI position weight matrix (PWM) developed from single base-pair profiling experiments 14,16 (Fig. 1B) to predict cleavage sensitivity.
Potential new SHS identified and assessed by the above criteria were then rank-ordered and experimentally validated by PCR amplification and mCreI in vitro cleavage analyses. Site-specific primer pairs were designed using CLC Workbench Primer Design Tool (http://www.clcbio.com; CLC Bio, Boston, MA) to generate ~300-400 bp PCR products containing the mCreI target site (Table S1).
Genomic DNA purified from human 293T cells using a Wizard Genomic DNA Purification Kit (Promega, Madison, WI) was used as the template for SHS amplifications (Table S1) glycerol, 150 mM EDTA) prior to electrophoresis through a 1% agarose gel run in TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA). Substrate and cleavage product bands were identified following gel electrophoresis by ethidium bromide staining, digital image capture and band intensity quantification using ImageJ (http://imagej.nih.gov/ij/). A comparably-sized PCR product containing the native mCreI target site was included in experiments as a positive digestion control. A subset of newly identified SHS were also sequence-verified from PCR products using SHS-specific primers by capillary sequencing (Table S1; Genewiz, South Plainfield, NJ). Sequenced reads were aligned to genomic sequence using CLC Workbench Alignment tool (CLC Bio, Boston, MA).
We verified the in vivo cleavage sensitivity of several potential SHS by co-expressing the mCreI homing endonuclease together with the TREX2 3ʹ to 5ʹ repair exonuclease in 293T cells. The inclusion of TREX2 allows a more accurate measure of the fraction of sites cleaved in vivo by promoting NHEJ-mediated mutagenic repair following site cleavage 22 (Fig. S1). The expression vector used in these experiments was constructed in a pRRL-based lentiviral vector backbone that encoded the open reading frames for mCreI, the TREX2 exonuclease and mCherry fluorescent protein in a single translational unit separated by self-cleaving T2A peptides 25 (Fig. S1). Target site cleavage was estimated by amplifying sites from transfected cells, then determining the fraction of PCR products that were mCreI cleavage-resistant and mutant. We extensively analyzed three new SHS in this way: SHS231, a unique chromosome 4 site with the highest SHS score; SHS229, a chromosome 2 SHS with perfect nucleotide sequence identity to a member of our 20 bp site query library; and SHS253, the chromosome 2-specific member of the small family of 6 identical target sites represented once each on 6 different chromosomes (chromosomes 2, 5, 7,14,17 and X; Fig. 1C, Table 2).
A modified calcium phosphate (CaPO 4 ) transfection protocol 23 was used to introduce a pRRL-based lentiviral expression vector encoding mCreI, TREX2 and mCherry proteins into human 293T cells 24 (Fig.  S1). Cells (2-4 x 10e5/well) were plated in a 6-well plate 24 hr prior to transfection and were ~70% confluent at the time of transfection. Expression vector plasmid DNA (1.5 µg in 10 µL H 2 O) was mixed with 40 µL of freshly prepared 0.25 M CaCl 2 and 40 µL of 2x BBS buffer (50 mM BES pH 6.95 (NaOH), 280 mM NaCl, 1.5 mM Na 2 HPO 4 ; Boston BioProducts), then incubated at room temperature for 15 min before being added dropwise to wells. Plates were incubated overnight in 3% CO 2 at 37°C. The medium was changed the following day, and cells were grown for an additional 24 hr in a 5% CO 2 , 37°C humidified incubator. Transfection efficiency was checked by determining the fraction of mCherrypositive cells by flow cytometry: in brief, cells were trypsinized, counted and fixed with formaldehyde (1% v/v final concentration, 10 min at room temperature followed by the addition of 1/20 volume of 2.5 M glycine) prior to flow cytometric analysis of ~2 x 10e4 cells/transfection on a BD FACS Canto II flow cytometer (BD Biosciences, San Jose, CA). Genomic DNA prepared from co-transfected and control cells was used for PCR amplification and in vitro mCreI cleavage analysis of specific SHS as described above.

Homology-dependent SHS editing by three genome engineering nucleases
The mCreI-I expression vector described above, together with SHS231-specific TALEN and CRISPR/Cas9 expression vectors, were used for SHS editing experiments. The SHS231-specific TALEN protein pair was designed using the TALEN Targeter 2.0 web design engine 26,27 ( https://tale-nt.cac.cornell.edu/node/add/talen ). A SHS231-specific CRISPR/Cas9 expression vector was constructed in pX260 29,30 that contained expression cassettes for the S. pyogenes Cas9 nuclease, the CRISPR RNA array, and the tracrRNA.
In order to determine whether SHS cleavage in vivo could catalyze homology-directed repair in the presence of a homologous donor template, we co-transfected human 293T cells with a SHS-specific repair template and an expression vector for mCreI, for a TALEN pair, or for Cas9 cleavase/nickase enzymes (Fig. 2, Fig. S1). The template for SHS-specific, homology-dependent repair consisted of 500 bp homology arms that flanked the mCreI target site region and contained a 48 bp insert at the center harboring a canonical loxP recombinase site and adjacent, diagnostic restriction endonuclease cleavage sites for PvuI and SacII (Fig. 2). Repair templates were made by overlap extension PCR using oligonucleotide primers to generate PCR products that, when re-amplified, incorporated the 48 bp loxP insert at the center of the repair template (Table S1).
Calcium phosphate transfection (as described above) was again used to introduce nuclease expression vectors into human 293T cells 24 . Transfection efficiency was checked by determining the fraction of mCherry-positive cells by flow cytometry, as described above. Molecular characterization of SHS editing was performed by PCR amplifying the SHS region of interest from transfected cells, followed by PvuI or SacII restriction digest to confirm targeted integration of the loxP cassette (Fig. 2,   Fig. S2). PCR products were also cloned into a pGEM-T Easy plasmid vector (Promega, Madison, WI) and transformed into α-Select Chemically Competent Gold Efficiency cells (Bioline, Taunton, MA), followed by plasmid preparation from white (insert-containing) colonies for capillary sequencing using a T7 promoter sequencing primer (Fig. 2). Sequencing results were aligned with the repair template sequence using the CLC Main Workbench software (CLCBio).

Homology-independent SHS genome editing by Cas9
Homology-independent editing of the SHS231 locus was performed using the protocol above with modified Cas9 and repair template constructs. Dual human U6-driven guide RNAs (gRNA) targeting SHS231 were simultaneously inserted into a custom S. pyogenes Cas9-T2A-GFP expression plasmid (pUS2-SH231) using Gibson assembly, as previously described 31 . SHS231-specific gRNAs (SHS231 gRNA1: 5'-GCCTCCCCCATAGTACCAT-3' ; SH231 gRNA2: 5'-G ATGTGCTCACTGAGTCTGA-3' ) were designed to target and cleave both the SHS231 genomic locus and the repair template to promote efficient transgene integration by NHEJ-mediated DNA end joining 32,33 . The transgene cassettes were also flanked by Bxb1 recombinase and ΦC31 attP integrase target sites that, once integrated, could be used for high efficiency SHS-specific editing by these recombinase/integrase proteins.

Stable gene expression from SHS231 transgene insertions
Transgene stability following SHS231 integration was analyzed by selection and GFP expression (Fig. 4A ) . Time-course imaging of GFP fluorescence was performed using an EVOS imaging system (Life Technologies), and the continued expression of SHS231 transgene-encoded Cas9 was quantified by qRT-PCR SYBR green fluorescence on an CFX96 quantitative PCR (qPCR) machine (Cas9 qFwd; The efficiency of SHS231 editing by different endonucleases was determined by co-transfecting two independent RMS cells lines (SMSCTR and RD) with a puromycin-expressing SH231 repair template along with an expression vector for mCreI, for Cas9 nickase (with a single gRNA), or for Cas9 cleavase (with single and dual gRNAs). The RMS cells were also co-transfected with the SHS231 repair template and piggybac transposase plasmid (PB210PA-1, Palo Alto, CA), to compare the SHS231 knockin efficiencies of mCreI and transposase-mediated transgene integration. Two days following transfection, cells were plated into 24 well plates at 3 x 10e4 cells/well, followed by growth in the presence of puromycin (2.5 µ g/ml) for 10 days. Cells were then fixed with 2% paraformaldahyde, stained with 0.5% crystal violet and imaged on a Nikon SMZ-745 stereomicroscope to quantify cell number by counting crystal violet stained pixels using imageJ software (NIH).

New human safe harbor site identification
Our BLAST search of 128 predicted highly cleavable mCreI target site variants revealed 27 unique mCreI target sites matches in the human genome ( Fig. 1A and 1B). A majority of these target sites were found only once (24/27, 89%), while the remaining 3 were represented 2, 3 or 6 times in the human genome for a total of 35 target site matches at different genomic locations (Fig. 1C, Table 2).
One of these target sites was a perfect match to a mCreI target site variant (a 20/20 bp match, or 100% identity), whereas the other hits differed by 1 bp (i.e., were 19/20 bp matches or 95% identical) to a query site sequence. The 35 mCreI target sites were located on 16 of the 23 human chromosome pairs including the X chromosome, and covered nearly half of all chromosome arms (23 of 48; Fig. 1C, Table   2).
All 35 new target sites, together with the three canonical human SHS AAVS1 , CCR5 and hROSA26 , were next evaluated using 8 safety, functional and accessibility criteria in addition to site uniqueness (Table 1 and 2). Among our 35 newly identified sites, 25 (or 71%) fulfilled more than half (≥5/9) of our SHS criteria, as did the AAVS1 and CCR5 canonical human SHS (Table 2). When we examined safety criteria alone (SHS criteria 1-6 in Table 1), 21/35 (60%) of our target sites met ≥4 of 6 criteria, with three (SHS231, 233 and 303) matching all 6 safety criteria. In contrast, the widely used human SHS AAVS1 , CCR5 and hROSA26 each matched only 3 of 6 safety criteria ( Table 2). This site assessment was more extensive than previous attempts and made systematic use of genomic data that together, allowed us to rank-order both newly identified and canonical SHS for potential utility and experimental verifications ( Table 2).
Genetic variation between individuals has the potential to complicate or disrupt the editing of SHS as well as other genomic regions. In order to assess the potential magnitude of this problem, we assessed all 35 of our new SHS for copy number and basepair-level genetic variation. None of our target sites was located in a copy number-variable region of the human genome, though we did identify predicted to strongly suppress mCreI cleavage efficiency by ≥70% (Fig. 1B, Table S2). Of note, among individuals analyzed as part of the 1KGP, 80% lacked any SNP variants in any of our 35 target sites including SHS231, and 94% had all 35 target sites predicted fully mCreI-cleavage sensitive despite the presence of one or more permissive base-pair variant SNP (Table S2 and additional results not shown).

Experimental validation of potential new human SHS
In order to experimentally validate the most promising of our potential new SHS, we amplified 28 of the target site regions from the human genome and subjected these to either in vitro mCreI cleavage assays or DNA sequencing. As part of these analyses we identified one polymorphic 108 bp insertion adjacent to SHS231 that was present in a subset of human cell lines. This insertion contained a 35-base poly-T sequence and adjacent short sequence blocks reminiscent of transposable element short tandem duplications, and was found to be an exact match for a segment of an AluYa5 subfamily, SINE-derived repeat of 311 bp that is present in ~4000 non-redundant copies in the human genome  (Table S2; additional results not shown).

Efficient in vivo cleavage and editing of new SHS by multiple genome editing nucleases
We assessed the functional competence of potential new SHS by determining their in vivo cleavage sensitivity and ability to be edited by different genome editing nuclease/repair template combinations.
These experiments focused on the single copy, highly-ranked chromosome 4q SHS231, and two sites on chromosome 2 that were single copy (SHS229), or as a single copy on chromosome 2 with additional copies on chromosome arms 5p, 7q, 14q, 17q and Xp (SHS253; Fig.1, Table 2). The in vivo cleavage sensitivity of these and three additional SHS was analyzed by co-expressing mCreI with the TREX2 3ʹ to 5ʹ repair exonuclease in human 293T cells, followed by PCR amplification and mCreI digestion of target sites. This experiment was designed to identify a cleavage-resistant target site fraction in nuclease-expressing cells, from which a minimum estimate of in vivo cleavage efficiency can be derived 22 .
Five of the 6 SHS assayed in this way, the unique sites SHS227, 229 and 231 and copies of the same target site sequence located on different chromosomes (SHS253, 257 and 263), had increased fractions of mCreI-resistant target site PCR products that ranged from 3.8% to 31.3% when compared with the corresponding SHS-specific PCR product from mock-transfected control cells. The presence of multiple SHS-specific, mCreI-resistant PCR products also provides evidence for the ability of mCreI to cleave-and thus potentially simultaneously edit-multiple target sites in human cells.
In order to determine whether SHS cleavage in vivo could catalyze high fidelity homologydependent repair, we co-transfected human 293T cells with an expression vector for mCreI, for a CRISPR/Cas9 cleavase/nickase or for a TAL effector nuclease (TALEN) pair together with a SHS-specific repair template containing a loxP site flanked by two different diagnostic restriction sites (Fig. 2). SHS229, 231 and 253 were analyzed following mCreI expression, SHS229 and 231 after CRISPR/Cas9 cleavase/nickase expression, and SHS231 after TALEN expression. PCR amplicons from transfected cells were then subjected to PvuI and SacII restriction digestion to confirm targeted capture and site-specific integration of the loxP repair template, followed by cloning and DNA sequencing to confirm the structure and fidelity of cleavage-dependent, targeted SHS integration (Fig.   2). The frequency of targeted SHS231 integration events in 293T cells was 4.8% for mCreI/TREX2 (3/63 clones); 6.1% (2/33) for CRISPR/Cas9 nuclease and 16.1% (5/31) for CRISPR/Cas9 nickase; and 1.23% (1/81) for a SHS231-specific TALEN pair (Fig. 2). Infrequent single base substitutions observed in cloned and sequenced loxP inserts were most likely PCR errors introduced by Taq DNA polymerase during site amplifications for cloning and DNA sequencing. Parallel targeted integration assays at SHS229 and 253 showed comparable results (Fig. S2).
In order to increase SHS engineering efficiency and potentially facilitate the editing in post mitotic cells, we also evaluated SHS231 editing by a potentially homology-independent knockin approach. This strategy used Cas9-mediated cleavage of the repair template and genomic SHS target locus (i.e., using dual gRNAs; US2-Cas9) to promote potential repair with transgene integration by NHEJ-mediated repair mechanisms 32,33 (Fig. 3A). While indel mutations can be introduced during NHEJ-mediated repair in the cleaved target locus and repair template, this is not a serious concern since our SHS were specifically identified to contain no functional genomic elements and the repair template cleavage site did not inactivate the encoded transgene(s). Molecular analysis of SHS231 integration events by amplification, cloning and sequencing of the 5' SHS231 integration site identified both direct fusion events (no indels), as well as the expected short indel mutations at the gRNA cleavage site (Fig. 3A), evidence compatible with NHEJ-mediated integration. The efficiency of dual gRNA Cas9 cleavage-mediated editing of the SHS231 locus was compared to the Cas9 nickase, cleavase and mCreI-mediated HDR approaches by co-transfection of each endonuclease with a repair template expressing puromycin (Fig. 3B-C, Fig. S1). The efficiencies of these endonucleases was also compared to random integration of the repair template using a piggybac transposon, since the repair template contained piggybac terminal repeat sequences flanking the transgene cassette. This experiment was performed in two independent RMS cells lines (RD and SMSCTR), where the putative homology-independent insertion or knockin of the puromycin repair template was 2-fold higher when compared to HDR-mediated insertion. Neither of these approaches, however, was as efficient as random integration by piggybac-mediated transposition ( Fig. 3B and 3C).

Characterization of stability, expression, and functionality of SHS231 integrated genes
The functional utility of any SHS depends critically upon persistent marking and/or SHS-specific gene expression after site editing. In order to assess this key SHS functional requirement, we analyzed the expression of several different transgene cassettes that had been integrated into the chromosome 4 SHS231. SHS transgene expression stability was assessed by integrating, and then following the expression of, a SHS231 GFP reporter cassette in two independent RMS cells lines (SMSCTR and Rh5) where transgene insertion was mediated by putative homology-independent editing. When GFP transgene expression was followed over several weeks (i.e., over 45 days) in the absence of antibiotic selection, we observed no significant decrease in GFP expression after 15 population doublings (Rh5) or 25 population doublings (SMSCTR; Fig. 4A). These results highlight the stable nature of transgene integration and expression from SHS231, over usefully long periods of time in mitotically dividing cells.
We next determined whether SHS231-integrated, Cas9-derived transgenes were not only persistently expressed but retained their intended functions. Stable Cas9-expressing cell lines are a convenient starting point for a growing range of Cas9-enabled methods to study gene structure, function or to enable genetic screens. We observed readily detectable Cas9 expression from SHS231 knockin transgenes that was comparable to cells super-infected with high titer lentivirus to express Cas9 protein, or to the expression of endogenous GAPDH protein (Fig. 4B). The functional competence of SHS231-expressed Cas9 protein was further demonstrated in Rh30 RMS cells by transducing cells with a lentivirus expressing two gRNAs targeting a PAX3/FOXO1 fusion oncogene contained in Rh30 (Fig. 4C). Efficient generation of the predicted 17,188 bp gDNA-targeted deletion in PAX3/FOXO1 was readily detected by PCR amplification of gRNA-transduced cell pools using primers that flanked the PAX3/FOXO1 gRNA target sites (Fig. 4C).
In a third series of SHS functional validation experiments, we integrated transgene cassettes in SHS231 that expressed chimeric Cas9-derived transcriptional activators dCas9-VPR or Cas9-VPR by Cas9-mediated knockin. VPR is a tripartite transcription factor consisting of VP64, P65 and Rta transactivation domains 34 . Fusion of this transcription factor to the C-terminus of the Cas9 protein generates a potent, programmable transcriptional activator (dCas9-VPR or Cas9-VPR) 34 . Each SHS231 RMS cell line expressing dCas9-VPR or Cas9-VPR was then transduced with a lentivirus expressing 2 or 3 gRNAs targeting the promoter region of the MYF5 gene (Fig. 4D). MYF5 is typically not expressed or expressed at very low levels in many RMS cells, and therefore is a good candidate for measuring gRNA-targeted Cas9-VPR-mediated gene activation. We found that both full length (20bp) and truncated (14 bp) gRNAs promoted robust Cas9-VPR-dependent MYF5 gene activation in both of the RMS cell lines tested (Fig. 4D).
These results collectively demonstrate efficient editing of a newly defined human safe harbor site, and the stable expression of functionally useful SHS231-integrated transgenes encoding GFP and Cas9 protein variants. Moreover, we demonstrate the ability of these proteins to drive additional useful outcomes including genome editing with the promotion of large deletions in a PAX3/FOXO1 fusion oncogene, and induced expression of the MYF5 gene that is normally silent in RMS cells. The SHS231-specific targeting vectors used in these experiments have been assembled into a SHS231-specific 'toolkit' to enable facile editing of the highly-ranked SHS231 in a wide range of human cell types (Fig. S1, Table S3). This SHS231 toolkit is available from Addgene (Addgene, Cambridge, MA), and includes both Cas9 and dCas9-based expression cassettes, as well as GFP and RFP reporter constructs with puromycin, hygromycin and blasticidin selectable markers. All of the expression vector transgenes included in this set are driven by the human EF-1α promoter and contain additional attP sites to serve as 'landing pads' for ΦC31 and Bxb1-mediated, high efficiency SHS transgene insertion.

Discussion
Only a small number of SHS are in wide use in human cells. These were originally identified by serendipity ( AAVS1 , CCR5) or by their similarity to SHS in other organisms (e.g., hROSA26) . In order to address the continuing need for additional well-validated human SHS to enable a broader range of basic and translational science applications, we used a systematic approach to identify and evaluate 35 potential new SHS in the human genome. These new SHS cover a substantial fraction of the human genome: 16 of 23 chromosomes including the X chromosome, with SHS on 23 of 48 chromosome arms ( Figure 1). These potential new SHS were assessed and rank-ordered as potential 'safe harbors' using both previously suggested criteria (e.g., 17 ) and additional more recently available human genome-scale structural, genetic and regulatory data (e.g., ENCODE data 18 ). Over half of our new SHS (20/35, or 57%) met 4 of our 6 core safety criteria (Tables 1 and 2), in contrast to the widely used human AAVS1 , CCR5 and hROSA26 SHS that each met 3 or fewer of these core safety criteria ( Table 2).
All 35 of these newly identified SHS contained a site-anchoring 20 bp mCreI nuclease cleavage site, and thus can be immediately targeted either singly or in multiplexed fashion using this small, easily vectorized homing endonuclease together with SHS-specific repair templates [7][8][9]                , and Cas9 targeting was performed using a common guide RNA and either a Cas9 cleavase or nickase, Numbers to the right of each row indicates the number of independent targeting events that were cloned and sequenced (see text for additional detail).