🖥️ BIOINFORMATICS OF CRISPR CAS9 | Designing guide RNAs (gRNAs) for Genome Editing | Adwoa Biotech
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READ ME: At
READ ME: At 1:07 when I mention that the most common cas is CAS9, I'm referring to the laboratory setting and not the biological CRISPR Cas systems in nature.
The crispr cas9 system was discovered as a result of observations in the mid 2000s. During this time Researchers who were studying bacterial sequences noticed that there were these repetitive palindromic sequences. So the palindromic sequences would then have these unique sequences that were flanked by these palindromic sequences. so the unique sequences were found to be representatives of viral genomes that the bacteria would keep if and when it survived an infection. so a bacteria that had a crispr system would keep these snippets of viral genomes and flank it with these repetitive sequences, if and when it survived an infection by a particular virus. And so it turns out that these are adaptive immune responses from bacteria. These repeats would be processed and it would then associate with another RNA molecule before it can then bind this DNA cutting enzyme known as CRISPR associated and so its given this 'CAS' and it has a number, the most common one is cas9 but you can have different cas (CRISPR associated) enzymes which are able to cut DNA. So let's give some terminology that you encounter with CRISPR Cas9 systems. So you come across Cas9, and Cas9 as we know, these CRISPR associated enzymes can cut DNA. They can recognise particular DNA sequences - when the guide RNA guides it. So then there's this guide RNA concept. So the guide RNA is this RNA that's used in bacterial adaptive responses. It's adapted for use in genome editing. So in the bacteria its two separate RNA components but in the lab when it's used as a tool, it's combined into a single RNA, guide RNA (sgRNA) that's about 100 base pairs of sequence that directs Cas9. So cas9 binds to it, it binds to this guide RNA (gRNA) and the guide RNA in turn, binds to one strand of double-stranded DNA. The first 20-21 base pairs of the guide RNA (gRNA) is what gives it specificity as to which DNA segment its going to bind. And that's where you get to program it and tell cas9 the 20 base pairs in your genome that you want cas9 to bind to. And then cas9 bound to that will edit that sequence. 3 base pairs from the PAM closest to that sequence.
So i'm going to show these examples using Sickle Cell Anemia as the genetic disease model that we may want to intervene in by designing guide RNA (gRNA) to fix the genetic defect.
So let's have a look at how we'd go about something like that.
The first step is to go to NCBI entrez and there you can search for the gene that's responsible for the disease. the sickle cell disease comes about where in the haemoglobin subunit beta you have 2 thymine (nucleotides) instead of adenine (nucleotides).
Once you obtain the gene sequence from NCBI, you can then look at structure of the gene and annotate where the exons are. The exons are the portions of the gene that will be retained in a mature messenger RNA transcript. To put it another way, the sequence that will be translated into the protein that you are studying. Within the exons, annotate the start codon, all exons, any untranslated regions (UTRs) and stop codon. This will help you to know which sequences are the best candidates for designing your guide RNA against.
GUIDE RNA DESIGN
Firstly, look at the gene and identify Protospacer Adjacent Motifs (PAMS). For Cas9, the PAM is NGG
The RNA guide for cas9 needs to be 20 nucleotides that are immediately next to the PAM on the 5' (upstream) side.
A more practical approach to designing/selecting the guide RNA (sgRNA) is to use a software such as ChopChop. Oligonucleotide manufactures such as Synthego also offer gRNA selection/design tools on their website.
Website where you can post gene sequences to remove numbers, spaces, get complementary sequences and or inverted sequences, making the sequences more searchable: http://www.thelabnotebook.com/sequenc...
Credit for palindromic sequence image: https://en.wikipedia.org/wiki/Palindr...
Sickle Cell Anemia video credit: https://www.ted.com/talks/amber_m_yat...
//
This video is about designing gRNA for crispr cas9 editing, genome editing with crispr cas9, bioinformatics of crispr cas9 genome editing, guide RNA design for cas9, cas9 editing sgRNA design, sgRNA design, guide RNA design, bioinformatics of cas9, cas9 bioinformatics, crispr cas9 bioinformatics, genome engineering with cas9, CRISPR, designing CRISPR/Cas guide RNA, designing guide RNAs for crispr cas9
.
// ADWOA
I'm Adwoa (Adwoa Biotech), a Biotechnology graduate. I’ve worked in medical research for years and want to be useful to people new to the lab life. This channel takes you through some of the techniques and concepts I've learnt working as a Research Assistant. Hopefully it helps if you're new to the topic/technique.
The crispr cas9 system was discovered as a result of observations in the mid 2000s. During this time Researchers who were studying bacterial sequences noticed that there were these repetitive palindromic sequences. So the palindromic sequences would then have these unique sequences that were flanked by these palindromic sequences. so the unique sequences were found to be representatives of viral genomes that the bacteria would keep if and when it survived an infection. so a bacteria that had a crispr system would keep these snippets of viral genomes and flank it with these repetitive sequences, if and when it survived an infection by a particular virus. And so it turns out that these are adaptive immune responses from bacteria. These repeats would be processed and it would then associate with another RNA molecule before it can then bind this DNA cutting enzyme known as CRISPR associated and so its given this 'CAS' and it has a number, the most common one is cas9 but you can have different cas (CRISPR associated) enzymes which are able to cut DNA. So let's give some terminology that you encounter with CRISPR Cas9 systems. So you come across Cas9, and Cas9 as we know, these CRISPR associated enzymes can cut DNA. They can recognise particular DNA sequences - when the guide RNA guides it. So then there's this guide RNA concept. So the guide RNA is this RNA that's used in bacterial adaptive responses. It's adapted for use in genome editing. So in the bacteria its two separate RNA components but in the lab when it's used as a tool, it's combined into a single RNA, guide RNA (sgRNA) that's about 100 base pairs of sequence that directs Cas9. So cas9 binds to it, it binds to this guide RNA (gRNA) and the guide RNA in turn, binds to one strand of double-stranded DNA. The first 20-21 base pairs of the guide RNA (gRNA) is what gives it specificity as to which DNA segment its going to bind. And that's where you get to program it and tell cas9 the 20 base pairs in your genome that you want cas9 to bind to. And then cas9 bound to that will edit that sequence. 3 base pairs from the PAM closest to that sequence.
So i'm going to show these examples using Sickle Cell Anemia as the genetic disease model that we may want to intervene in by designing guide RNA (gRNA) to fix the genetic defect.
So let's have a look at how we'd go about something like that.
The first step is to go to NCBI entrez and there you can search for the gene that's responsible for the disease. the sickle cell disease comes about where in the haemoglobin subunit beta you have 2 thymine (nucleotides) instead of adenine (nucleotides).
Once you obtain the gene sequence from NCBI, you can then look at structure of the gene and annotate where the exons are. The exons are the portions of the gene that will be retained in a mature messenger RNA transcript. To put it another way, the sequence that will be translated into the protein that you are studying. Within the exons, annotate the start codon, all exons, any untranslated regions (UTRs) and stop codon. This will help you to know which sequences are the best candidates for designing your guide RNA against.
GUIDE RNA DESIGN
Firstly, look at the gene and identify Protospacer Adjacent Motifs (PAMS). For Cas9, the PAM is NGG
The RNA guide for cas9 needs to be 20 nucleotides that are immediately next to the PAM on the 5' (upstream) side.
A more practical approach to designing/selecting the guide RNA (sgRNA) is to use a software such as ChopChop. Oligonucleotide manufactures such as Synthego also offer gRNA selection/design tools on their website.
Website where you can post gene sequences to remove numbers, spaces, get complementary sequences and or inverted sequences, making the sequences more searchable: http://www.thelabnotebook.com/sequenc...
Credit for palindromic sequence image: https://en.wikipedia.org/wiki/Palindr...
Sickle Cell Anemia video credit: https://www.ted.com/talks/amber_m_yat...
//
This video is about designing gRNA for crispr cas9 editing, genome editing with crispr cas9, bioinformatics of crispr cas9 genome editing, guide RNA design for cas9, cas9 editing sgRNA design, sgRNA design, guide RNA design, bioinformatics of cas9, cas9 bioinformatics, crispr cas9 bioinformatics, genome engineering with cas9, CRISPR, designing CRISPR/Cas guide RNA, designing guide RNAs for crispr cas9
.
// ADWOA
I'm Adwoa (Adwoa Biotech), a Biotechnology graduate. I’ve worked in medical research for years and want to be useful to people new to the lab life. This channel takes you through some of the techniques and concepts I've learnt working as a Research Assistant. Hopefully it helps if you're new to the topic/technique.
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