TABLE 3 |.

Troubleshooting

Step	Problem	Possible reason	Possible solution
9 (B, ix)	Absence of colonies	Incompatible overhangs	Check that the overhangs in the sgRNA cloning oligonucleotides are correct
9 (B, ix)	Colonies growing on negative control plate	Incomplete digestions of pCRL1 or equivalent plasmid	Perform overnight digestion of the plasmid; increase amount of BsaI; treat the digested plasmid with phosphatase to reduce self-ligation
9 (B, xii)	No sgRNA sequences	Incomplete digestion of cloning plasmid; ligation failure	Screen additional colonies; re-digest the plasmid; check the oligos sequences
21	No cleavage or low cleavage rate of PCR amplicon containing the intended target site	Impure input PCR product; sgRNA for the particular locus does not mediate cleavage; Cas9 lost activity	Purify PCR product; re-design sgRNA; check Cas9 activity with another amplicon and sgRNA
73	Poor library enrichment	Adapter dimers that compete with the adapter-ligated DNA during the PCR	Perform DNA size-selection of the adapter-ligated/USER treated DNA fragments using PippinHT