Bstxi

To determine the efficiency of CRISPR/Cas9 to induce site-specific mutations in injected larvae, we used restriction digest assays (Supplemental Figure 2). CRISPR guides were designed to target sequences containing restriction digest sites, such that when indels were introduced by DNA repair, the restriction site is disrupted. Genomic DNA was extracted from individual larvae at 2 dpf. Larvae were placed individually in 0.2 ml PCR tubes in 90 μl 50 mM NaOH and boiled at 95° for 20 min. Ten μl Tris-HCl pH 8 was added as a reaction buffer and mixed thoroughly. RT-PCR using Firepol® (Solis BioDyne) was used to amplify a 235 bp region (for cxcr4b) and a 259 bp region (for cxcl12a) around the PAM site. Gene specific primers were designed using the Primer 3 web tool (http://primer3.ut.ee/). Primer sequences were as follows: cxcrb4_fwTCCCGTATACTGTAGGGAGGAcxcr4b_revTTTTTGCATTTTGTTTTCTTGcxcl12a_fwTTCTCTGTGGGACTGTGTTGACcxcl12a_revTTCGAAAATTTGACCCAAAAGT. Restriction enzyme digests were then performed using bsII at 55° for 2 h (for cxcr4b) and bstXi (New England Biolabs) at 37° for 2 h (for cxcl12a). Products were run using gel electrophoresis on a 2% gel (Supplemental Figure 2).

Strong sgRNAs against essential genes or cell surface markers were selected from the hCRISPRi-v2 library (Horlbeck et al., 2016a (link); Nuñez et al., 2021 (link)). Intermediate-activity sgRNAs were selected either from the hCRISPRi-v2 library or by incorporating defined mismatches in strong sgRNAs (Jost et al., 2020 (link)). All sgRNA sequences used for individual evaluation are listed in Supplementary file 8.
Individual sgRNA expression constructs were cloned as described previously (Gilbert et al., 2014 (link)). Briefly, two complementary oligonucleotides (IDT), containing the sgRNA targeting region as well as overhangs matching those left by restriction digest of the vector with BstXI and BlpI, were annealed and ligated into pCRISPRia-v2 (pU6-sgRNA EF1Alpha-puro-T2A-BFP with two SbfI sites flanking the sgRNA expression cassette, Addgene #84832; Horlbeck et al., 2016a (link)) or pU6-sgRNA EF1Alpha-puro-T2A-mCherry (a gift from Gregory Ow and Eric Collisson, UCSF; Jost et al., 2020 (link)) digested with BstXI (NEB or Thermo Fisher Scientific) and BlpI (NEB) or Bpu1102I (Thermo Fisher Scientific). The ligation product was transformed into Stellar chemically competent Escherichia coli cells (Takara Bio) and plasmid was prepared following standard protocols. The resulting sgRNA expression vectors were individually packaged into lentivirus as described above.

REs (NcoI, BtgI, XbaI, NdeI, NheI, BmtI, BamHI, XcmI, PflMI, BstEII, NcoI, HpaI, BbsI, BsgI, AfeI, BstXI, StuI, BsrGI, and EcoRI, accompanied with respective buffers) were from New England Biolabs (NEB). Another NdeI (accompanied with H buffer), and recombinant DNase I (RNase-free) were from Takara-Bio. Another NcoI (accompanied with H buffer) and DNase I were from Nippon Gene. Hi-Lo DNA marker was from Bionexus. DNA primers were synthesized by Thermo Fisher Scientific. Other chemicals were from FUJIFILM Wako Pure Chemical Corporation unless otherwise noted.

Individual sgRNA expression vectors were cloned by first annealing complementary synthetic oligonucleotide sequences containing each sgRNA protospacer (Table S3) flanked by BstXI (New England Biolabs) and BlpI (New England Biolabs) restriction sites (Integrated DNA Technologies). Each double-stranded annealed pair was then ligated into a BstXI/BlpI-digested pCRISPRi-v2 expression vector containing a BFP cassette. Each sgRNA expression vector was then packaged into lentiviruses in HEK 293T Lenti-X cells and were individually transduced into either two or three color CEBPA CRISPRi reporter lines at a MOI < 1, resulting in ~20–30% infected cells by BFP expression. Cells were sorted on BFP 2 days post-transduction to select for sgRNA expression and allowed to recover in RPMI 1640 for 6 days. Reporter expression was then measured by flow cytometry.