Fastdigest esp3i

For Cas9-based library, lentiGuide-Puro (Addgene#52963) were digested with FastDigest Esp3I (Thermo Fisher Scientific) and Gibson Assembly was used to clone Cas9-based spacer into the backbone. For AsCpf1-based library, site-directed point mutagenesis was used to incorporate 2 Esp3I (BsmBI) sites flanking Cas9’s guide scaffold, generating lentiUniversal-Puro. LentiUniversal-Puro then were digested with FastDigest Esp3I (Thermo Fisher Scientific) and Gibson Assembly was used to clone AsCpf1 mono-cistronic guide into the backbone. For multiplexed AsCpf1 library, guide arrays were cloned into the same backbone with Quickligase ligation kit (NEB). DH10B MegaX (Life technologies) electroporation competent cells were used for transformation. Lenti-X 293 T(Clonetech) were transfected with plasmid library, PMD2.G(Addgene #12259), PsPAX2 (Addgene #12260) to generate lentiviral libraries.

All 71,090 gRNAs were synthesized as 58-mer oligonucleotides on one microarray chip (Custom Array) and amplified by PCR as a pool. The PCR products were purified using QIAquick nucleotide removal kit (Qiagen) and cloned into a modified version of the all-in-one lentiviral vector lentiCRISPRv2 (Addgene), which contains the fSpCas9 gene. The lentiCRISPRv2 vector was digested with FastDigest Esp3I (Thermo Fisher Scientific) and treated with shrimp alkaline phosphatase (NEB) for 30 min at 37°, heat-inactivated for 10 min at 65°, and gel-purified using the QIAquick Gel Extraction kit (Qiagen). Using a one-step digestion and ligation reaction, purified library PCR pool was cloned into the digested lentiCRIPSRv2 vector at a ratio of 1:5 vector-to-insert molar ratio. The ligation reaction was precipitated using Pellet Paint Co-Precipitant (EMD Millipore) and 1 µl of the precipitated ligation was transformed into Endura ElectroCompetent cells (Lucigen). To yield a 1200-fold representation of the library, 10 identical ligation reactions were pooled and purified followed by 40 parallel transformations. Outgrowth media from transformations were pooled and plated onto 100 15-cm LB-carbenicillin (100 µg/ml) plates. Colonies were scraped off plates, pooled, and the plasmid DNA was extracted using the QIAfilter Plasmid Mega kit (Qiagen).

The cDNA sequence encoding sgRNA which targets a conserved sequence in exon 7 of human DNMT3A gene was synthesized and subcloned into LentiCRISPR-v2 plasmid (Addgene 52961, kindly provided by Dr. Jian Huang at Temple University, Philadelphia, PA) to make the lentiDNMT3A-sgRNA-Cas9 construct. Briefly, the forward and reverse primers including 20 bp target DNMT3A sequence and BsmbI sticky ends were annealed and inserted into the LentiCRISPR-v2 plasmid digested with FastDigest Esp3I (Thermo Fisher Scientific, #FD0454) (Fig. 1b). sgRNA primer sequences have been reported by Gundry MC et al. previously and were shown in Table 1 [22 (link)].Fig. 1

Schematic diagram of sgRNA targeting DNMT3A. a The structure of DNMT3A gene and the three common transcripts. Black vertical lines: exons. Horizontal lines: introns. Arrow: the location of sgRNA targeting exon 7. b The structure of lentiCRISPR v2 plasmid. The arrows indicate the sgRNA sequence