5) CRISPR Cas9 - Screening and Validation Strategies

Summary

This video explains the workflow for CRISPR screening and various methods to validate gene editing. It details the process of creating indels using Cas9, describing the four potential outcomes in a diploid cell and the desired biallelic or homozygous mutations. The video outlines screening strategies like mismatch cleavage assays and amplicon sequencing for polyclonal pools, followed by serial dilution and monoclonal validation. Sanger sequencing and Next Generation sequencing are recommended for thorough analysis, and the video also highlights the importance of checking for off-target effects. Overall, it provides a comprehensive guide for creating a validated CRISPR knockout cell line.

Takeaways

• 😀 Successful CRISPR experiments can be validated by screening the cell line for specific edits.
• 😀 Indels caused by non-homologous end joining can result in frameshift mutations, knocking out a gene.
• 😀 There are four possible outcomes when editing a diploid cell: no edit, heterozygous, biallelic, and homozygous mutations.
• 😀 Biallelic or homozygous mutations are ideal for ensuring the gene is completely knocked out.
• 😀 The first step in CRISPR screening is to assess the polyclonal pool using mismatch cleavage assays or PCR amplicon sequencing.
• 😀 Mismatch cleavage assays use nucleases like Surveyor or T7E1 to detect mismatches in DNA strands due to editing.
• 😀 PCR amplicon sequencing (Sanger or Next Generation) can also be used to identify editing in the polyclonal pool.
• 😀 Next Generation sequencing provides high sensitivity, quantitative data, and sequence information, but is more expensive and complex.
• 😀 After confirming editing in the polyclonal pool, single cells are isolated and expanded to form monoclonal cell lines.
• 😀 For monoclonal validation, Sanger sequencing or Next Generation sequencing can be used to check for biallelic or homozygous mutations.
• 😀 High-throughput Next Generation sequencing can screen many monoclonal cell lines in parallel, and whole genome sequencing helps detect off-target effects.

Q & A

What is the main purpose of CRISPR screening?

The main purpose of CRISPR screening is to identify whether a specific gene has been successfully edited or knocked out in a cell line by using various validation methods like mismatch cleavage assays, Sanger sequencing, or Next Generation sequencing.

What are indels in the context of CRISPR editing?

Indels, or insertions and deletions, occur when a double-stranded DNA break created by Cas9 is repaired via the non-homologous end joining pathway, resulting in small insertions or deletions at the target site. These can lead to a frameshift mutation, knocking out the gene.

What are the possible outcomes when targeting a single gene in a diploid cell?

The possible outcomes when targeting a single gene in a diploid cell are: 1) no edit occurs, 2) one allele is edited (heterozygous mutation), 3) both alleles are edited with different sequences (biallelic mutation), or 4) both alleles are edited with the same sequence (homozygous mutation).

Why are biallelic or homozygous mutations typically desired?

Biallelic or homozygous mutations are typically desired because they ensure the gene of interest is completely knocked out, which is important for accurate functional analysis of the gene.

What is the first step in the CRISPR screening workflow?

The first step in the CRISPR screening workflow is to screen the polyclonal pool of cells to check if any editing has occurred, using methods like mismatch cleavage assays, Sanger sequencing, or Next Generation sequencing.

How does the mismatch cleavage detection assay work?

The mismatch cleavage detection assay involves PCR amplification of the target region, denaturation and reannealing of DNA strands to form heteroduplexes if editing occurred, followed by treatment with nucleases like T7E1 or Surveyor nuclease that cleave mismatched DNA strands. Cleavage bands indicate editing.

What are the advantages and disadvantages of using Sanger sequencing for CRISPR screening?

Sanger sequencing is a sensitive method for detecting editing, but it has limitations in analyzing quantitative data or overlapping traces from edited polyclonal pools. It's often sufficient to confirm whether editing occurred, but detailed sequence analysis can be challenging.

Why might Next Generation sequencing be preferred over Sanger sequencing for CRISPR screening?

Next Generation sequencing is preferred over Sanger sequencing because it is highly sensitive, quantitative, and provides detailed sequence data, allowing for a more comprehensive analysis of CRISPR edits. However, it is more expensive and complex to analyze.

What is the purpose of isolating single cells after CRISPR screening?

Isolating single cells after CRISPR screening allows for the creation of monoclonal cell lines, which can be further screened to identify a cell line with the desired genetic edit, such as a biallelic or homozygous mutation.

What is the typical method for monoclonal validation using Sanger sequencing?

The typical method for monoclonal validation using Sanger sequencing involves PCR amplification of the targeted region from each monoclonal cell line, cloning the amplicons into vectors, and sequencing them. The presence of frameshift mutations in all sequences indicates successful gene knockout.