It depends on what you're doing...  If you have an obvious size difference, then just do PCR and resolve the products by gel electrophoresis.  If there's one product, it's homozygous.  If there are both products, it's heterozygous.  

You should strive for this situation.  For example when making a knockout, it's best to eliminate a large sequence with two independent cuts, or to insert a long sequence with stop codons, selectable marker and all.  This will not just simplify your testing, but also make your life easier in other ways (no spontaneous reversion / selectability...) 

If you must make a deletion too short to see by PCR and standard gel electrophoresis, then you can sequence the PCR products.  The mutations will show up on a clean Sanger trace.  WT and KO traces will be overlayed -- so the mutated bases show up as mix-base peaks at half the height.  So make sure you get a high quality sequence, and they give you the raw traces. 

sequencing

Thanks everyone.

Yeah. I wanted to do T7 analysis, and just wanted to know if it is possible to say that the assay result can be mono or bi allelic. Of course, need to sequence still for confirmation. 

My another question is how to differentiate homozygous from bi allelic or di allelic. still learning these things....

You can do high resolution melting curves using EVA Green or another saturating dye, with this method it is very easy to distinguish between mono or biallic events, additionally you get a first hint about the size and the kind of mutation and you also identify chimeric events. Very cool method ;) which works excellent for A. thaliana

I do not have a paper in mind but just search for high resolution melting analysis I think it was established by Gundry ? et al., 2003 ?

IN GENERAL:

1. If you have a diploid plant with AA genotype for a special trait, and you design a gRNA to target 'A' allele, the CRISPR/Cas system could cause mutagenesis on both 'A' alleles for the homologous chromosomes. Because both 'A' alleles' sequences are the same. [biallelic knockout]

2. If you have a diploid plant with AA genotype for a special trait, and you design a gRNA to target 'A' allele, the CRISPR/Cas system could cause only one 'A' allele to muate, instead of two 'A's.  [monoallelic knockout]

That sounds really clear to me Yuan-Yeu Yau. 

But if I only targeted the "a" in "Aa". Can I still have the same mutation in "A" in the next generation? OR to have bi-allelic in the progenies.

Hi Christian,

1. I saw you are from IRRI, dealing with rice and assuming that the plants you are working on are highly homozygosity nearly all loci. The genotype of the trait you are working on is, for example, BB, and when you apply your CRISPR/Cas, you could either get a mono-allelic knockout (BB') or bi-alleleic knockout (B'B', this is what we desire).

2. If you are using transformation on plants, many reports now show that biallelic mutants (B'B') generated by CRISPR/Cas9 technology can be obtained in the primary (T0)  transformants

Can you please explain the PCR method of finding out if the clone you generated is mono allelic vs bi allelic. Thanks

@ Saswati,

1.  Let's say you want to knockout a trait of a rice plant with genotype AA. After CRISPR/Cas9 treatment, your plants can have AA' (mono-allelic mutation) or A'A' (bi-allelic mutation). A: normal allele, A': mutant allele. If the A' is mutated by a large deletion/insertion, then you can design primers from A for PCR to distinguish the 3 genotypes (AA, AA' and A'A'). The PCR results: 1 band for AA and A'A' (biallelic mutation), and two bands for AA' (monoallelic mutation). The band sizes for AA and A'A' are different, due to large deletion and insertion in A' allele.

However, As John mentioned above, quite often the deletion (or indel) and insertion observed in A' are quite small (ex. 1 bp or 2 bp), in this case, it is almost impossible to distinguish A' from A by observing the PCR band sizes on a gel.

@ Saswati,

Now there is another way to distinguish monallelic mutation from biallelic mutation [from Takara]:

See "Successful Identification of Monoallelic and Biallelic Mutants after CRISPR/Cas9 Gene Editing" (see attached PDF)

http://www.clontech.com/US/Products/Genome_Editing/CRISPR_Cas9/Technical_Notes/Identification_of_Monoallelic_and_Biallelic_Mutants

After you obtained those monoallelic and biallelic mutants, you can set up an in-vitro cleavage reaction, using the same sgRNA used for the initial gene editing to cleave.

You can proceed as suggested by Dr. Yuan-Yeu Yau above. I have worked as technical support scientist for Clontech for several years and the results of the above kit are highly efficient. Just follow the protocol as described.

Good Luck

consider this paper for help

Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9 https://www.nature.com/articles/nature17664

Determining whether a mutation introduced by CRISPR/Cas9 is monoallelic (affecting only one allele) or biallelic (affecting both alleles) involves a few clear steps:

1. PCR Amplification: Amplify the targeted region using PCR. This will generate a fragment encompassing the expected mutation site.

2. Sanger Sequencing: Perform Sanger sequencing on the PCR product. A monoallelic mutation typically results in a clean sequence up to the mutation site, followed by overlapping peaks (indicative of heterozygosity). In contrast, a biallelic mutation will show a clean sequence with the mutation present and no overlapping peaks after the mutation site.

3. T7 Endonuclease I Assay: This assay exploits mismatches in heteroduplex DNA. If both alleles are different, the enzyme will cut the DNA at the mismatch site, resulting in two bands on an agarose gel. If the mutation is biallelic, no cut will occur, resulting in a single band.

4. Restriction Fragment Length Polymorphism (RFLP): If the mutation creates or abolishes a restriction enzyme site, RFLP can be used to differentiate between monoallelic and biallelic mutations based on the pattern of DNA fragments after restriction enzyme digestion.

5. Digital Droplet PCR (ddPCR) or Quantitative PCR (qPCR): These techniques can be used to quantify the mutation. A biallelic mutation will have a higher proportion of mutant alleles compared to a monoallelic mutation.

6. Clonal Isolation and Screening: Isolate single cells, grow them into clones, and screen each clone for the presence of the mutation. This will tell you if one or both alleles in each clone carry the mutation.

By combining these methods, you can accurately determine the zygosity of mutations created by CRISPR/Cas9.

Take a look at this protocol list; it could assist in understanding and solving the problem.