CRISPR-mediated ADNP Knockout and Knockin

To generate ADNP knockout, knock in, and homeodomain deletion cell lines with CRISPR/cas9, design of guide RNAs was carried out using the CRISPR Design Tool (https://zlab.bio/guide-design-resources) and inserted into PX459, a gift from Feng Zhang (Addgene plasmid: 62988)^{77 (link)} or into lentiCRISPRv2 Blast, a gift from Brett Stringer (Addgene plasmid: 98293)^{78 (link)}. To generate pCDNA3-ADNP donor plasmid, gBlock gene fragments were synthesized (IDT) and inserted into pCDNA3 using NEBuilder. To generate ADNP knockout cell lines for CRISPRa system, guide RNAs were inserted into CRISPRa-sgRNAs were designed according to Konermann et al.^{64 (link)} and inserted into pLentiV2-dCas9-VP64, a gift from Igor Ulitsky (Addgene plasmid: 141104). 1 µg guide RNAs and 1.5 µg donor plasmid were transfected into mESC cell line. ADNP knock in and homeodomain deletions candidate clones were confirmed by PCR using the extracted DNA that was isolated using QuickExtract (Epicentre QE09050), and further confirmed using western blot. All primer sequences can be found in Supplementary Table 1. All cell lines generated in this study are available upon request.