CRISPR

Question 1

CRISPR

Answer 1

Class 2 Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) systems, which form an adaptive immune system in bacteria, have been modified for genome engineering.

Engineered CRISPR systems contain two components: a guide RNA (gRNA or sgRNA) and a CRISPR-associated endonuclease (Cas protein). The gRNA is a short synthetic RNA composed of a scaffold sequence necessary for Cas-binding and a user-defined ∼20 nucleotide spacer that defines the genomic target to be modified. Thus, one can change the genomic target of the Cas protein by simply changing the target sequence present in the gRNA.

Question 2

CAS

Answer 2

CRISPR associated proteins

Question 3

what’s is CRISPR

Answer 3

it is a prokaryotic adaptive immune system that allow bacteria to defend against invading genetic element

Question 4

Stages of CRISPR

Answer 4

1) adaptation or spacer acquisition: A phage infect the bacteria which leads to a sequence of the invading DNA called photospacer to be incorporated into the CRIPSR array by Cas protein.

2) crRNA biogenesis: the CRIPR array have to be transcribed, which create a long precursor CRISPR RNA (full length). this pre cr RNA start to form these loop which are important for the complex. It is then process into a mature CRIPSR RNA, each encoding a unique spacer sequence and contains tandem repeats from the bacteria that recognize cas9.

3: target interference: upon new infection, the mature crRNA will bind the to the invading DNA sequence based on complementarity and near a PAM sequence (NGG spent only on foreign DNA), which allow foreign DNA cutting by the Cas effector proteins (endonuclease- (eats DNA from the end).

Question 5

Cripsr array

Answer 5

are identical repeats which are interspaced by phage derived spaces
spacers are unique

Question 6

precRNA processing

Answer 6

tracRNA interacts with each repeat sequence to generate a dsRNA, along with Cas9 help
dsRNA is then cleave by RNAase 3 which liberates crRNA from the precursor to form the gRNA. The 5 ‘ end is further processed to form the guide sequence of 20 nt

Question 7

targeting process

Answer 7

Cas 9 scans the target DNA for a region of complementarity with the guide CrRNA. Once found, it introduce a dsDNA break.For cleavage however, a PAM sequence is necessary immediately downstream of the target site

Question 8

PAM

Answer 8

photospacer adjacent motif

only present in target DNA of phage, never in bacterial DNA

Question 9

Non homologous end joining repair

Answer 9

You introduce a deletion by ds break. NHEJ is a cell repair machinery that is error prone. It will repair the dsDNA by leaving indells, leading to loss of function (66%) or gain of function (33%). loss of function wanted - you need to target the n terminal.

Question 10

Homology directed repair

Answer 10

HDR is another repair mechanism not such efficient in mammalian cell. this require the addition of a donor template. sometime used to add GFP expression.
It is repaired via homologous recombination with a donor template, not very efficient but it has a high fidelity.

Question 11

a gRNA should be

Answer 11

is a scaffold sequence for Cas binding and it define the genomic target

20 nt long
upstream of PAM immediately
unique
specific

Question 12

experiment plan

Answer 12

design and construct your crisp CAS component
transfect cells to bring lentiviral expression with you plasmid
take supernatant and transduce the cells of interest
study the effect

Question 13

Generating a Knockout Using CRISPR

Answer 13

you can use CRISPR to generate knockout cells or animals by co-expressing an endonuclease like Cas9 or Cas12a (also known as Cpf1) and a gRNA specific to the targeted gene. The genomic target can be any ∼20 nucleotide DNA sequence, provided it meets two conditions:

1) The sequence is unique compared to the rest of the genome.
2) The target is present immediately adjacent to a Protospacer Adjacent Motif (PAM).

The PAM sequence serves as a binding signal for Cas9, but the exact sequence depends on which Cas protein you use.

We’ll use the popular S. pyogenes Cas9 (SpCas9) as an example, but check out our list of additional Cas proteins and PAM sequences. Once expressed, the Cas9 protein and the gRNA form a ribonucleoprotein complex through interactions between the gRNA scaffold and surface-exposed positively-charged grooves on Cas9. Cas9 undergoes a conformational change upon gRNA binding that shifts the molecule from an inactive, non-DNA binding conformation into an active DNA-binding conformation. Importantly, the spacer region of the gRNA remains free to interact with target DNA.

Cas9 will only cleave a given locus if the gRNA spacer sequence shares sufficient homology with the target DNA. Once the Cas9-gRNA complex binds a putative DNA target, the seed sequence (8-10 bases at the 3′ end of the gRNA targeting sequence) will begin to anneal to the target DNA. If the seed and target DNA sequences match, the gRNA will continue to anneal to the target DNA in a 3′ to 5′ direction. Thus, mismatches between the target sequence in the 3′ seed sequence completely abolish target cleavage, whereas mismatches toward the 5′ end distal to the PAM often still permit target cleavage.

Cas9 undergoes a second conformational change upon target binding that positions the nuclease domains, called RuvC and HNH, to cleave opposite strands of the target DNA. The end result of Cas9-mediated DNA cleavage is a double-strand break (DSB) within the target DNA (∼3-4 nucleotides upstream of the PAM sequence).

The resulting DSB is then repaired by one of two general repair pathways:

The efficient but error-prone non-homologous end joining (NHEJ) pathway
The less efficient but high-fidelity homology directed repair (HDR) pathway
The NHEJ repair pathway is the most active repair mechanism, and it frequently causes small nucleotide insertions or deletions (indels) at the DSB site. The randomness of NHEJ-mediated DSB repair has important practical implications, because a population of cells expressing Cas9 and a gRNA will result in a diverse array of mutations (for more information, jump to Plan Your Experiment). In most cases, NHEJ gives rise to small indels in the target DNA that result in amino acid deletions, insertions, or frameshift mutations leading to premature stop codons within the open reading frame (ORF) of the targeted gene. The ideal end result is a loss-of-function mutation within the targeted gene. However, the strength of the knockout phenotype for a given mutant cell must be validated experimentally.

Question 14

Knock in of point mutation

Answer 14

you try to introduce a precise and specific mutations in the gene of interest

you first cut in the genomic locus with a single guide RN targeting the desired site
you then provide the repair template which carry the desired mutation and a stretch of homologous overlap with the region of interest in the genome
you then allow the repair thorough HDR

Question 15

gene tagging

Answer 15

trying to make a gene fusion of an endogenous gene with a tag of choice with as fluorescence

you cut at the genomic locus with a specific sgRNA
you provide a PCR product with homologous ends
you allow repair of the genomic cut via integration (MMEJ) of the donor to field an in frame fusion with the tag

Question 16

MMEJ

Answer 16

micro homology mediated end repair
it allow the gene tagging of larger DNA sequence integration

Question 17

Genome-Wide Screens Using CRISPR

Answer 17

the ease of gRNA design and synthesis, as well as the ability to target almost any genomic locus, make CRISPR the ideal genome engineering system for large-scale forward genetic screening. Forward genetic screens are particularly useful for studying diseases or phenotypes for which the underlying genetic cause is not known. In general, the goal of a genetic screen is to generate a large population of cells with mutations in, or activation/repression of, a wide variety of genes and then use these cells to identify the genetic perturbations that result in a desired phenotype.

Before CRISPR, genetic screens relied heavily on shRNA technology, which is prone to off-target effects and false negatives due to incomplete knockdown of target genes. In contrast, CRISPR is capable of making highly specific, permanent genetic modifications that are more likely to ablate target gene function. CRISPR has already been used extensively to screen for novel genes that regulate known phenotypes, including resistance to chemotherapy drugs, resistance to toxins, cell viability, and tumor metastasis. Currently, the most popular method for conducting genome-wide screens using CRISPR involves the use of pooled lentiviral CRISPR libraries.

What are pooled lentiviral CRISPR libraries?

Pooled lentiviral CRISPR libraries (often referred to as CRISPR libraries) are a heterogenous population of lentiviral transfer vectors, each containing an individual gRNA targeting a single gene in a given genome.

Guide RNAs are designed in silico and synthesized (see panel A below), then cloned in a pooled format into lentiviral transfer vectors (panel B). CRISPR libraries have been designed for common CRISPR applications including genetic knockout, activation, and repression for human and mouse genes.

Each CRISPR library is different, as libraries can target anywhere from a single class of genes to every gene in the genome. However, there are several features that are common across most CRISPR libraries. First, each library typically contains ∼3-6 gRNAs per gene to ensure modification of every target gene, so CRISPR libraries contain thousands of unique gRNAs targeting a wide variety of genes. gRNA design for CRISPR libraries is usually optimized to select for gRNAs with high on-target activity and low off-target activity, and libraries may use different algorithms for gRNA design.

Keep in mind that the exact region of the gene to be targeted varies depending on the specific application. For example, knockout libraries often target 5′ constitutively expressed exons, but activation and repression libraries will target promoter or enhancer regions. Be sure to check the library information/original publication to see if a library is suitable for your experiment. Libraries may be available in a 1-plasmid system, in which Cas9 is included on the gRNA-containing plasmid, or a 2-plasmid system in which Cas9 must be delivered separately.

Although lentiviral libraries containing Cas9 are the most popular method for CRISPR screening, they are not suitable for all cell types or experiments. Mammalian CRISPR libraries have also been created in AAV backbones for in vivo experiments and in a retroviral backbone for delivery to cells that are poorly transduced by lentivirus. Non-mammalian CRISPR libraries are also available. Additionally, although CRISPR has been less widely used in bacteria due to technical challenges, several bacterial CRISPR libraries have been developed for inhibition using dCas9.