Off-Target Effects in CRISPR Gene Editing
Learn about off-target effects in CRISPR gene editing, caused by misbinding of gRNA with non-target DNA, leading to additional mutations and genomic instability. Explore strategies like extending PAM-sequence for higher specificity and the use of dCas9 mutants to minimize off-target effects while leveraging CRISPR advantages.
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Presentation Transcript
Off-Target Effects by CRISPR Pascal Kr ger Gene Editing by CRISPR Johanna-Wittum-School Pforzheim
Off-target effects Off-target effects are caused by misbinding of the gRNA with non- target DNA Off-target effects cause additional mutations in the genome These can lead to genomic instability For example, different gRNA-structures can have a different influence on the DNA These target-sites are then split into on and off-target sites
Off-target effects 17 bp gRNA + Cas9 20 bp gRNA + Cas9 3 NGG 5 NGG Double- stranded DNA CRISPR/Cas9 Off-target cut CRISPR/Cas9 On-target cut 3 5 On-targetarea Off-target area
Length and specificity The PAM-sequence activates Cas9 to cut the DNA target sequence The PAM-sequence for Cas9 is NGG N means a position on target DNA which is variable and could be A, T, C or G PAM could be AGG, TGG, GGG CGG
Length and specificity of the PAM-sequence Extending the PAM-sequence to 5 bp reduces the number of possible binding sites This increases the specificity This happens because of mathematics: 43 = 64 45 = 1024 A longer PAM-sequence increases the specificity of the CRISPR/Cas- complex and therefore ensures fewer binding sites
What is the advantage of a longer PAM-sequence? The extension of the PAM-sequence ensures a higher specificity of the CRISPR/Cas-complex The extension reduces the possible off-target effects The longer PAM-sequnce can reduce the number of off-target effects
The use of dCas9 dCas9 = dead Cas9 Dead Cas9 is a mutant of Cas9 dCas9 can only lead to a single strand break There are also dCas9 mutants that no longer have any cutting function dCas9 is used, for example, to bring transcription factors to a desired gene location This way, one can use the advantage of CRISPR/Cas9 without actually cutting the target sequence
The use of dCas9 Two dCas9 proteins are required to induce a double-strand break using dCas9 Both also need one gRNA structure two in total A slightly offset double-strand break occurs When an off-target cut occurs, only a single-strand DNA break occurs These are easy and quick to repair
gRNA + dCas9 20 bp gRNA + dCas9 5 NGG 5 dCas 9 single-strand break Double-stranded DNA 3 3 NGG dCas 9 single-strand break 20 bp gRNA + dCas9
How do you design a gRNA? In science, programs are used that design gRNA for the planned CRISPR/Cas9 cut In the program you can also see the statistical probability of off- target effects Since each gRNA is programmable, you have to enter various information about the planned cut in the program
gRNA-Design with the help of CRISPOR First: you type in to the program, the sequence you want to use This sequence can not be longer then 2300 bp It has to be an exon-sequence Our sequence is 600 bp long Second: you select the target genome
gRNA-Design with the help of CRISPOR In the second step you choose the PAM sequence you want to use The program has a pool of PAM sequences These are composed of gRNA+PAM sequence
gRNA-Design with the help of CRISPOR The program indicates the length of the entered sequence The cut is expected at -3 bp from the PAM sequence You now get a list of the most effective gRNA sequences
gRNA-Design with of CRISPOR The program indicates all statistically possible off-target effects In our most effective gRNA, there is a possible off-target effect of 4 mismatches This is a very good gRNA Can also look different
gRNA-Design with CRISPOR (0-0-0-0-14) 1 missmatches = 100% match 2 missmatch = 100% match 3 missmatches = 100% match 4 missmatches = 100% match 5 missmatches = 14 Off-target-effects If 16 bases on the genome bind with the gRNA, 14 possible off-target effects can arise
