So you’ve done your CRISPR experiment! Now, how can you tell if your cell line was successfully edited? In this video, we’ll discuss the workflow for CRISPR screening as well as different validation methods After Cas9 has created a double-stranded DNA break at the target region Repair via the non-homologous end joining pathway can result in the creation of short insertions or deletions called indels. If an indel results in a frameshift mutation for all copies of a gene, that gene will have been knocked out When targeting one gene of a diploid cell, there are 4 possible outcomes 1) no edit occurs. 2) One allele is edited. This is called a heterozygous mutation. 3) both alleles are edited but they carry a different sequence. This is called a biallelic mutation. or 4) Both alleles are edited and they carry the same sequence. This is called a homozygous mutation. In most cases, a biallelic or homozygous mutation is desired in order to be sure the gene of interest is completely knocked out. The basic workflow for CRISPR screening is 1) Screen your polyclonal pool to see what proportion of cells was edited using the mismatch cleavage assay Sanger amplicon sequencing or, Next Generation amplicon sequencing. 2) If some of the population was edited, isolate single cells, then expand to create monoclonal cell lines. 3) Screen monoclonal cell lines for one which has the desired edit using Sanger sequencing, or, Next Generation sequencing After performing your CRISPR experiment The first step is to screen your polyclonal pool of cells to see if any editing occurred We recommend screening your polyclonal pool using either the mismatch cleavage detection assay, or PCR amplicon sequencing. The mismatch cleavage detection assay can be performed using either Surveyor nuclease or, T7 endonuclease 1. These specialized nucleases create a double-stranded break at the site of a mismatch between two different strands of annealed DNA. The mismatch cleavage detection assay consists of four steps 1) The edited region is PCR amplified 2) The DNA strands are denatured then, reannealed. The strands of DNA will separate then randomly re-hybridize, creating heteroduplexes if editing has occurred. 3) DNA is treated with T7E1 or Surveyor nuclease. DNA will be cut only if the strands have formed a heteroduplex. 4) The cleaved DNA is run on an agarose gel. If cleavage bands are present, a portion of the pool has been edited. The brighter the cleavage products the more efficient the editing was. The mismatch cleavage detection assay is a quick and inexpensive method to test polyclonal pools for editing. Amplicon sequencing can be done using Sanger sequencing or Next Generation sequencing. In either case the target region is amplified by PCR, first. Sanger sequencing of a PCR amplicon is a more sensitive detection method, but it can be difficult to analyze for quantitative or sequence information. This is because sequencing reads for an edited polyclonal pool will have many overlapping traces downstream of the target site. These overlapping traces cannot be distinguished with typical Sanger sequencing analysis programs. But their presence or absence is usually enough to determine whether any editing has occurred in the pool. Next Generation amplicon sequencing may also be used for polyclonal screening. This technique has a few advantages over others for CRISPR screening. 1) It’s extremely sensitive. 2) It’s quantitative. 3) It provides sequence data. However, this method is much more expensive and complicated to analyze than others. So, you’ve screened your polyclonal pool, and now know that it has undergone some editing. The next step is to perform serial dilution to isolate single cells from your pool These cells can be expanded to create monoclonal cell lines The next round of screening is done to find a cell line that has a biallelic or a homozygous mutation, creating a frameshift mutation. We recommend performing monoclonal validation using either Sanger sequencing or Next Generation sequencing as these will give you the most information about your monoclonal cell lines. For monoclonal validation by Sanger sequencing, the typical method is 1) PCR amplify the targeted region from each monoclone. 2) Clone the amplicons into a vector. 3) Sanger sequence. This way, each vector will contain only one gene copy which will generate a clean trace when sequenced. A gene is considered to be fully knocked out if all sequences from the monoclone show frameshift mutations. Next Generation sequencing can also be used for monoclonal validation. It has the advantage of being able to perform high-throughput screening of many monoclones in parallel. As well, whole genome sequencing is an excellent way to investigate any off target editing that may have occurred during your CRISPR experiment. And that’s it! After following these guidelines, you should now have a validated knockout cell line. While we’ve discussed some of the more popular CRISPR knockout screening methods, there are many others that have been developed. For more information, check out our knowledge base article about CRISPR screening and validation. Thanks for watching!