SUPPLEMENTARY MATERIALS AND METHODS

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GFP-plasmids containing two identical protospacers (both before and ... the corresponding TL1 or TL2 protospacer bearing linker was ligated to them using T4 ...
SUPPLEMENTARY MATERIALS AND METHODS

Vector constructions

SpCas9-gRNA expressing plasmids (targeting human and mouse genes and also containing TL1 and TL2 spacers) were generated from the px330 vector (Addgene # 42230, 1) according to Hanhui Ma et al. 2. Primers used for cloning of these constructs are listed in Supplementary Data.

The nuclease inactive SpCas9 (dCas9) expressing plasmid was generated from the px335 (Addgene #42335, 1) SpCas9 nickase vector by mutating His840 to Ala, thereby creating a nuclease inactive SpCas9. Partial sequence of SpCas9 from px335 was amplified in two fragments by PCR using Phusion polymerase. The first fragment was amplified from the ApaI cloning site to position 840, coding for His (with the primers dCas9-ApaI, containing an ApaI recognition sequence and dCas9-H840A-rev, containing the Ala coding triplet at position 840 and an Eco31I recognition site). The second fragment was amplified from position 840 corresponding to His to the EcoRI cloning site (with the primers dCas9-H840A-for, containing the Ala coding triplet at position 840 and an Eco31I recognition site and dCas9-EcoRI-BD, containing a “Body Double enzyme” (Esp3I) recognition sequence for generating EcoRI overangs by mutating the EcoRI recognition site 3). The px335 was cut with EcoRI and ApaI and the large fragment was purified from agarose gel. The PCR products were cut with ApaI and Eco31I enzymes and were ligated to the backbone with T4 ligase.

The in-cell cleavable GFP-vectors containing Rb or Ttn targets were constructed as follows. Rb or Ttn protospacer bearing linkers (Rb-InCell1-linker or Ttn-InCell1-linker) were cloned into PscI digested pEGFP-C1 (Clontech) plasmid, creating the pRb-InCell1 or pTtn-InCell1 plasmids, respectively. GFP-plasmids containing two identical protospacers (both before and after the GFP expression cassette) were created by ligation of a second Rb or Ttn protospacer bearing linker (Rb-InCell2linker or Ttn-InCell2-linker) to these plasmids after digestion by MluI and using T4 ligase, resulting in: pRb-InCell2 or pTtn-InCell2 plasmids, respectively.

The self-cleaving plasmids were prepared as follows. From the previously prepared pTL1 or pTL2 Cas9-gRNA plasmids the TL1 or TL2 gRNA expression cassettes (U6promoter-gRNA) were amplified by PCR using Q5 polymerase (with primers U6gRNAPCR-gibson-fwd, U6gRNAPCR-gibson-rev) and were inserted into Eco31I-digested pEGFP-C1 plasmid using Gibson Assembly Master Mix according to the manufacturer’s protocol. The resulted plasmids were digested by PscI, and the corresponding TL1 or TL2 protospacer bearing linker was ligated to them using T4 ligase to result the final pSc1 and pSc2 plasmids, respectively. The 10 kbp self-cleaving plasmid, pSc1-10k, was prepared by blunt-end ligation of a 5618 bp DNA fragment (sequence available in Supplementary Data) into the MluI site filled with Klenow fragment of the previously made self-cleaving plasmid pSc1. The self-cleaving plasmid containing a puromycin cassette, pSc1-puro, was prepared by PCR-amplifying the puromycin cassette (hPGK-Puro-SV40polyA) from pLKO.1puro plasmid (Addgene #8453, 4) using Q5 polymerase and primers Sc1purogibson-PCR-fwd and Sc1purogibson-PCR-rev and was integrated into Esp3I-linearized pSc1 vector by using Gibson Assembly Master Mix.

The self-cleaving plasmid containing the ecDHFR-derived degradation domain (pSc1-DD) was prepared as follows. From a previously made construct (pDD-GFP, sequence available in Supplementary Data) the GFP cassette with an N-terminally fused ecDHFR-DD was cloned to pSc1 plasmid between NheI and Bsp1407I restriction enzyme sites. The pDD-GFP plasmid was originally created from pBMN DHFR(DD)-YFP (Addgene #29325 5) .

The homologous recombination donor plasmid (pHR-PRNP) targeting the Prnp gene was constructed as follows. Two linkers were cloned into pEGFP-C1 plasmid: one, bearing two BpiI enzyme recognition sequences (BpiI-linker) into VspI and PscI sites and another, bearing two Esp3I recognition sequences (Esp3I-linker) into AdeI and MluI sites (thereby creating pHR-noArm). The 1000 bp long homologous arms (to regions before the start codon and after the stop codon of the prion protein coding sequence) were amplified from genomic DNA isolated from N2a cells by nested PCR, using Phusion polymerase. In the first PCR the HR arms were amplified (using primers prpStart-for, prpStart-rev, and prpStop-for, prpStop-rev). In the second PCR the HR arms were amplified from the previous PCR using Esp3I and BpiI recognition sequence bearing primers (prpStart2-for, prpStart2-rev and prpStop2-for, prpStop2-rev, respectively) and were cloned to the previously generated GFP-plasmid (pHR-noArm) into the corresponding Esp3I or BpiI sites. From this construct the CMV promoter was eliminated by digesting the plasmid with BshTI and PscI, then it was 5’ filled with Klenow fragment and finally it was circularized with T4 ligase to result pHR-PRNP.

The Rosa26 locus targeting homologous recombination donor plasmid (pHR-ROSA) was constructed in a similar way to the PRNP-targeting HR donor plasmid. The 800 bp long homologous arms were amplified from pDonor-MCS-rosa26 plasmid (Addgene #37200, 6) using Phusion polymerase with primers bearing Esp3I and Bpi-sites (rosa1-for, rosa1-rev, and rosa2for, rosa2-rev) and were cloned into the previously prepared GFP-plasmid (pHR-noArm) with corresponding enzyme recognition sequences. In this plasmid the CMV promoter was not eliminated.

The pre-linearized GFP-plasmids (Figure 7 and Supplementary Figures S2, S9) were digested with the following enzymes: the once-cleaved plasmids with MlsI, the twice-cleaved ones with MlsI and MssI, and the three times-cleaved plasmids were digested with MlsI, MssI and EcoRV enzymes. The once in-cell cleavable plasmids on Figure 7 and Supplementary Figure S9 were pRb-InCell1 and pTtn-InCell1, whereas the twice cleavable were pRb-InCell2 and pTtn-InCell2.

The sequences of all plasmids and oligonucleotides described here are available in Supplementary Data.

SUPPLEMENTARY FIGURES

Supplementary Figure S1. The decay of transient transfection in NIH/3T3 cells

The percentages of the GFP positive cells measured at different days (between day 2 to 15) after cotransfection of a self-cleaving plasmid (pSc1) with an expression vector for various gRNAs (pten, p53, prp1-4, piwi2) and wild type SpCas9, are shown as colored lines. The various gRNAs are labelled on the figure by different colors and their names are shown from top to bottom corresponding to the decreasing order of the values measured at day 15. As controls, three conditions were used: one where the self-cleaving circular GFP-plasmid with the corresponding TL1 gRNA but without TL1 target was transfected along with an expression vector containing active SpCas9 and Rb target - showing the sum of the targeted and random integration of a circular plasmid (circular); a second, where a self-cleaving GFP-plasmid (pSc1) was co-transfected with an active SpCas9 and the corresponding TL1 gRNA expressing vector - showing the nontargeted, random integration of an in-cell opened plasmid (linear); and a third, where an inactive SpCas9 was used along with the circular self-cleaving GFP-plasmid - indicating random integration of a circular plasmid (dead). The values measured on day two were considered 100% for each case and those obtained on following days are normalized to the corresponding day 2 values.

Supplementary Figure S2. Genomic integration of a circular or a linear plasmid is not effective without homologous arms The percentages of cells harboring a stably integrated GFP-cassette after co-transfection by expression vectors for both SpCas9 and various gRNAs and a GFP-cassette-containing plasmid that was either circular (A) or linear (B, C) into either N2a (A, B) or NIH/3T3 cells (C). Various bars correspond to the various gRNAs used (prp1-3, sho and piwi1, light grey bars) that target three genes (Prnp, Sprn, Piwil4) with the sequences listed in Supplementary Data. As a control (dead, dark grey bars), an inactive mutant-SpCas9 was used for each case of cell type or GFP-plasmid type. Bars show the mean ± standard deviation (S.D.) of percentages measured in n=3 independent transfections, which were normalized to the transfection efficiency (% GFP positive cells measured on the 2nd day after transfection: ranging between 94-97%). The values were compared to that obtained for the control (dead). *: p