Primers used for construction of reporter plasmids.

<p>Primers used for construction of reporter plasmids.</p

Screening efficient ZFNs target goat alpha s1-casein gene via this yeast-based system.

<p>(<b>A</b>) <b>The first step screening for six 3-bp subsites.</b> Plasmids encoding randomized ZFN libraries were transformed into corresponding selection strains. And 10 µl transformants were plated on non-selective medium to test transformation efficiency and 100 µl transformants were plated on selective medium to obtain efficient ZFNs. LBS1(RBS1), LBS2(RBS2) and LBS3(RBS3) selection strains were transformed with appropriately randomized ZFN1, ZFN2 and ZFN3 libraries, respectively, to enrich fingers binding to three left (right) 3-bp subsites. (<b>B</b>) <b>Amplification of enriched 3 zinc fingers and assembly via overlap PCR.</b> Surviving colonies were scraped from selective plates, and plasmids encoding ZFNs were recovered from yeast. PCR was performed to amplify enriched fingers from enriched ZFNs plasmids. Left picture represents amplification of enriched 3 zinc fingers with 3-finger arrays targeting left half target site. Right picture represents amplification of enriched 3 fingers with 3-finger arrays targeting right half target site. Three individual fingers were assembled by overlap PCR with primers FF/FR. (<b>C</b>) <b>Screening for ZFNs target two half sites and the full length site.</b> LBS and RBS selection strains harboring left half site (5′-TAG GCT GTT-3′) and right half site (5′-GCA GTG AAC-3′) were transformed with re-constructed left and right ZFNs libraries, respectively. Plasmids encoding efficient ZFNHL and ZFNHR were recovered from survival yeast colonies on selective plates. FBS selection strain harboring full target site (5′-AAC AGC CTA TGATA GCA GTG AAC-3′) was co-transformed with ZFNHL and ZFNHR expression vectors. Plasmids encoding efficient ZFN pairs were recovered from yeast colonies on selective plates.</p

Unexpected binding behaviors of bacterial Argonautes in human cells cast doubts on their use as targetable gene regulators

<div><p>Prokaryotic Argonaute proteins (pAgos) have been proposed as an alternative to the CRISPR/Cas9 platform for gene editing. Although Argonaute from <i>Natronobacterium gregoryi</i> (<i>Ng</i>Ago) was recently shown unable to cleave genomic DNA in mammalian cells, the utility of <i>Ng</i>Ago or other pAgos as a targetable DNA-binding platform for epigenetic editing has not been explored. In this report, we evaluated the utility of two prokaryotic Argonautes (<i>Ng</i>Ago and <i>Tt</i>Ago) as DNA-guided DNA-binding proteins. <i>Ng</i>Ago showed no meaningful binding to chromosomal targets, while <i>Tt</i>Ago displayed seemingly non-specific binding to chromosomal DNA even in the absence of guide DNA. The observed lack of DNA-guided targeting and unexpected guide-independent genome sampling under the conditions in this study provide evidence that these pAgos might be suitable for neither gene nor epigenome editing in mammalian cells.</p></div

Primers used for construction of randomized ZFNs libraries.

<p>Primers used for construction of randomized ZFNs libraries.</p

The potential for high-precision targeting by pAgos.

<p>A. Targeting of CRISPR/Cas9 is limited by the requirement of a PAM site (green boxes). B. <i>Ng</i>Ago or <i>Tt</i>Ago might allow the precise targeting of features, such as transcription factor binding sites (black boxes). gRNA/gDNA, red lines.</p

Schematic diagram of yeast-based ZFNs screening and validation system.

<p>(<b>A</b>) <b>Schematic representation of simultaneous screening and validation of ZFNs in yeast.</b> Host strain AH109 harbored ZFNs expression and reporter plasmids and co-expressing ZFNs that could cut target sites on <i>Gal4</i> to generate double strand breaks (DSBs). DSBs were repaired via cellular single strand annealing (SSA) and <i>Gal4</i> was restored by removing the target site and one homology region. Thus, functional Gal4 started to drive expression of reporter genes in AH109, and yeast colonies survived on selective medium. By comparison, ZFNs had no abilities to cut target sites and dysfunctional <i>Gal4</i> could not drive reporter genes expression either. Therefore, yeast colonies could not survive on selective medium lacking histidine and adenine. Gal4HL: Gal4 homology left arm, Gal4HR: Gal4 homology right arm, Gal4AD: Gal4 active domain, Gal4DBD: Gal4 DNA binding domain. (<b>B</b>) <b>The procedure of screening efficient ZFNs via three-step selection.</b> The first step aimed at enriching efficient ZFNs target 3-bp subsites from randomized ZFNs libraries. 3 enriched single fingers target 9-bp half sites were amplified and assembled by overlap PCR and cloned into vector pLeu-FokI between <i>Xba</i>I/<i>Bam</i>HI sites to generate re-constructed ZFNs libraries. The second step screening aimed at screening for efficient ZFNs target 9-bp half sites from re-constructed ZFNs libraries. Finally, pairs of efficient ZFNs were obtained after the third step screening. RZF1: Enriched randomized-zinc finger 1, RZF2: Enriched randomized-zinc finger 2, RZF3: Enriched randomized-zinc finger 3.</p

h<i>Tt</i>Ago and h<i>Ng</i>Ago do not cleave genomic target sites.

<p>A. Schematic of human codon-optimized <i>Tt</i>Ago and <i>Ng</i>Ago proteins containing two nuclear localization signals (NLSs) and a 3xFlag epitope tag. Western blot analysis of h<i>Tt</i>Ago and h<i>Ng</i>Ago proteins in HEK293 cells using an antibody against the 3xFlag tag. Untransfected cells serve as a negative control (-). Ponceau staining was used as a loading control. B. Diagram illustrating <i>RPL13A</i> and <i>HER2</i> guide DNAs and genomic target site. Genomic target sites are indicated in blue and complementary ssDNA guides are indicated in red. C. Amplicon sequencing was carried out on HEK293 cells co-transfected with h<i>Tt</i>Ago or h<i>Ng</i>Ago expression plasmids with (+) or without (-) gDNAs to <i>RPL13A</i> and <i>HER2</i>. To increase the amount of gDNAs, cells were re-transfected with gDNAs 24 hours after the initial transfection (++). CRISPRESSO analysis confirms that h<i>Tt</i>Ago and h<i>Ng</i>Ago did not cause insertions or deletions (indels) under any of these conditions (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193818#pone.0193818.s008" target="_blank">S5 Table</a>). As a control, HEK293 cells were co-transfected with Cas9 nuclease and gRNA expression plasmids targeting <i>RPL13A</i> and <i>HER2</i>. RNA-guided Cas9 displayed target site cleavage at the genomic <i>RPL13A</i> and <i>HER2</i> target sites. The percentage of sequence reads containing indels relative to the total number of sequence reads is plotted on the y-axis.</p

Detection of ZFNs activities targeting endogenous locus in GME cells.

<p>(<b>A</b>) <b>Illustration of puromycin-based system for enrichment of genome modification-positive cells.</b> The reporter gene of <i>puromycin resistance</i> (<i>PuroR</i>) was divided into two fragments carrying 300-bp direct repeats and a ZFN target site. The target sites in reporter vectors as well as the genome could be cut by introducing ZFNs in GME cells. Thus, restored <i>PuroR</i> conferred GME cells resistance to high concentration of puromycin pressure. Meanwhile, genomes of surviving clones were also potentially targeted by ZFNs. Pcmv: CMV promoter, Puro-L: <i>PuroR</i> left homology, Puro-R: <i>PuroR</i> right homology, PAsv40: SV40 polyA. (<b>B</b>) <b>Schematic representation of T7 endonuclease I assay.</b> Genomic DNA was isolated from surviving clones and negative control. PCR reaction was used to amplify target sequences. Then, 200 ng PCR products were re-denatured and re-annealed to generate heteroduplexes. T7 EI enzyme specifically recognizes and cleaves mismatches, and cleaved fragments were isolated by 3% agarose gel. (<b>C</b>) <b>Levels of endogenous locus modification mediated by 3 pairs of ZFNs.</b> Goat mammalian epithelial (GME) cells were electroporated with reporter and ZFNs expression vectors or control plasmids. 5 µg/ml puromycin was added to culture 2 days post electroporation, and genomic DNA was isolated 5 days after treatment with puromycin. The T7 EI assay demonstrated that 3 pairs of ZFNs Z1, Z2 and Z3 generated gene disruption frequencies at 9.4%, 15.9% and 4.1%, respectively. No gene modification was detected in GME cells electroporated with control plasmids of empty expression vectors. (<b>D</b>) <b>Sequences of small deletions and insertions in target site of </b><b><i>α s1-casein</i></b><b> induced by ZFNs.</b></p

Construction of reporter vectors and randomized ZFN libraries.

<p>(<b>A</b>) <b>Schematic of constructing reporter plasmids.</b> Target sites with <i>Not</i>I/<i>Bam</i>HI sticky ends were generated via two oligonucleotides direct annealing (90°C 5 min, 70°C 10 min, cool down to room temperature), and inserted into plasmid pADH-Gal-MCS, resulting in reporter vector pADH-Gal4-BS. Reporter plasmids containing palindromic sequences of target sites for screening three 3-bp subsites of left (right) were substituted by BCR subsites, respectively. 9-bp half sites took place of BCR target sequence completely in LF (left half) and RF (right half) target sites. (<b>B</b>) <b>Illustration of constructing 3 randomized ZFN libraries.</b> The randomized finger 1 fragments were amplified with primers F1/F1R, finger 2 and finger 3 were amplified with primers F1D/FR and all 3 fingers were fused together by overlap PCR. PCR products were cloned in yeast expression plasmids pLeu-FokI between <i>Xba</i>I/<i>Bam</i>HI sites resulting in randomized ZFN1 library. The construction of randomized ZFN2 and ZFN3 libraries was similar to that of ZFN1 library. (<b>C</b>) <b>Sequence alignment results of randomized ZFN libraries.</b> The regions of red cube represent sequences of randomized zinc fingers in each library in which DNA sequences of key amino acids were mutated by PCR with randomized primers and the other two zinc fingers were fixed to the corresponding fingers of BCR-ZFP.</p

Summary of production of VDR gene modified mice via CRISPR/Cas9.

<p>Summary of production of VDR gene modified mice via CRISPR/Cas9.</p