Table 1:

Troubleshooting

Step	Issue	Possible reason	Solution
3	Gene ID not found in database	Gene is labeled differently in GRCz10 database	Use the transcript ID or Ensembl ID
4	No common sgRNAs targeting all isoforms of the gene	Several identified isoforms with differing lengths	Use a specific isoform as target; Use “Union” instead of “Intersection” on CHOPCHOP under Options → General → Isoform consensus determined by. The default setting on CHOPCHOP is “Intersection”, which searches for sgRNAs only in regions present in all isoforms of the gene. “Union” searches for sgRNAs in regions present in any isoform.
15	DNA template concentration low	Inefficient PCR assembly; Not enough templates	Redo the PCR reaction; Increase concentration of forward and reverse oligos
21	sgRNA concentration is low	Poor in vitro transcription	Repeat in vitro transcription using RNAse-free conditions; Set up multiple in vitro transcription reactions, combine during sample cleanup
23	sgRNA degraded	sgRNAs are susceptible to degradation by RNAses	Repeat in vitro transcription using RNAse-free conditions
28	DNA Barcode concentration is low	Inefficient PCR	Redo the PCR reaction; Set up multiple reactions, combine samples during cleanup
35	Droplets are not uniform	Clogged instrument	Do not use samples with high viscosity. Use recommended buffers
47	High backpressure during droplet injection	Excess oil in the needle	Use “clear’ to push out excess oil.
47	Oil is being accidentally injected in embryos (Supplementary Video 7)	High injection pressure; needle pore is too wide	Reduce injection pressure so that each droplet is injected in 2-3 foot pedal pushes; Ensure needle is trimmed to the right bore size.
47	Droplets have a lot of oil in-between	Excess oil in the needle	Use “clear’ to push out excess oil.
47	Droplets are too close to each other	High injection pressure; No oil in the needle	Reduce injection pressure so that each droplet is injected in 2-3 foot pedal pushes
47	Needle clogged (Supplementary Video 8)	Debris in the needle	Increase pressure to clear debris; Press “Clear”.
52	Low embryo survival	Poor injection technique; Unhealthy clutch of embryos	Optimize injection technique; Redo injection in a separate clutch
53	High incidence of gross deformity in injected embryos	Unhealthy clutch of embryos; High nucleic acid concentration	Redo injection in a separate clutch; Remeasure DNA barcode and RNA concentration; Reduce DNA barcode amount, if needed.
63	Negative control also shows barcode amplification	Ambient DNA barcode contamination	Setup barcode amplification reaction in a PCR workstation; Cleanup workstation after use
63	No barcode amplification	Barcodes have degraded; PCR amplification did not work	Perform barcode amplification at an earlier time point; Avoid adding tissue debris in PCR reaction; Set up a positive PCR control (DNA barcode spiked-in in lysate from un-injected sample)
66	Sanger sequencing results show overlapping signals	Multiple droplets injected in the embryo; Carryover contamination	Wash embryos thoroughly before lysis and DNA amplification
66	Poor sanger sequencing quality	Suboptimal DNA concentration	Submit the recommended amount for sequencing; Column cleanup samples before sequencing
71	“Hits” from screen do not show phenotype during secondary validation	Inefficient target editing during validation	Ensure efficient target gene editing; Re-inject droplets targeting the gene and assess phenotype
76	Phenotype is not rescued upon mRNA coinjection	Poor expression of the protein	Ensure the protein is being expressed using western blot of whole embryo lysate or fluorescent imaging (if tagged with a fluorescent marker)
76	Phenotype is not rescued upon mRNA coinjection	Phenotype is a result of off-target sgRNA activity	Inject individual sgRNAs in separate embryos and measure phenotype penetrance. If phenotype still observed, ensure targeted gene is being edited.