Lab Manual for Biomedical Research

Saturday, September 17, 2016

Researchers find way to boost CRISPR-Cas9 efficiency

SAN FRANCISCO, Aug. 28 -- Researchers at the University of California, Berkeley, have found a way to boost the efficiency of a gene-editing tool, known as clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR-Cas9), so that it cuts and disables genes up to fivefold in most types of human cells.

While the key to figuring out the role of genes or the proteins they code for in the human body or in disease is disabling the gene to see what happens when it is removed, CRISPR-Cas9 is the go-to technique for knocking out genes in human cell lines to discover what the genes do and holds the promise of accelerating the process of making knockout cell lines.

However, researchers must sometimes make and screen many variations of the genetic scissors to find one that works well.

In the new study, published in a the journal Nature Communications, the UC Berkeley researchers found that this process can be made more efficient by introducing into the cell, along with the CRISPR-Cas9 protein, short pieces of deoxyribonucleic acid (DNA) that do not match any DNA sequences in the human genome.

The short pieces of DNA, called oligonucleotides, seem to interfere with the DNA repair mechanisms in the cell to boost the editing performance of even mediocre CRISPR-Cas9s between 2½ and 5 times.

"It turns out that if you do something really simple - just feed cells inexpensive synthetic oligonucleotides that have no homology anywhere in the human genome - the rates of editing go up as much as five times," said lead researcher Jacob Corn, the scientific director of UC Bekeley's Innovative Genomics Initiative and an assistant adjunct professor of molecular and cell biology.

The technique boosts the efficiency of all CRISPR-Cas9s, even those that initially failed to work at all.

Corn portrays CRISPR-Cas9 gene editing as a competition between cutting and DNA repair: once Cas9 cuts, the cell exactly replaces the cut DNA, which Cas9 cuts again, in an endless cycle of cut and repair until the repair enzymes make a mistake and the gene ends up disfunctional.

Perhaps, he said, the oligonucleotides decrease the fidelity of the repair process, or make the cell switch to a more error-prone repair that allows Cas9 to more readily break the gene.

The next frontier, he was quoted as saying in a UC Berkeley news release, is trying to take advantage of the peculiarities of DNA repair to improve sequence insertion, in order to replace a defective gene with a normal gene and possibly cure a genetic disease.

Polymerase Chain Reaction (PCR) Analysis

PCR analysis is a technique that allows technicians to create millions of precise DNA replications from a single sample of DNA. In fact, DNA amplification alongside PCR can let forensic scientists perform DNA analysis on samples that are as tiny as only a couple of skin cells. In contrast to some other DNA analysis techniques, PCR analysis has the advantage of analysing minuscule sample sizes, even if they are degraded although they must not be contaminated with DNA from other sources during the collection, storage and transport of the sample.

DNA MICROARRAY PROTOCOL

i) Set-up the following Pre-Hybridisation solution in a Coplin Jar and incubate at65°C during the labeling incubation period to equilibrate. 20X SSC 8.75 ml 20% SDS 0.25 ml BSA (100 mg/ml) 5.0 ml H2O to 50.0 ml

ii) Label control and test genomic DNA as follows:- CONTROL TEST Genomic DNA ˜ 2 mg ˜ 2 mg Random Hexamers (3 mg/ml) 1 ml 1 ml H2O to 41.5 ml to 41.5 ml Heat at 95ºC for 5 minutes. Snap cool on ice and briefly centrifuge. 10X buffer 5 ml 5 ml dNTP's (5mM each dATP, dGTP & dTTP, 2mM dCTP) 1 ml 1 ml Cy-labelled dCTP 1.5 ml (Cy3) 1.5 ml (Cy5) Klenow fragment (10U/ml) 1 ml 1 ml Incubate at 37°C for 90 minutes.

iii) Incubate the microarray slide(s) in the Pre-Hybridisation solution for 20 minutes at65°C, beginning just before the end of the labelling reactions incubation time at37°C.

iv) Combine the control and test reactions and purify using the Qiagen MinElute PCR Purification kit, using a two step wash stage using 500 ml then 250 ml volumes of Buffer PE and eluting the labeled cDNA from the MinElute column with 14 ml H2O. The columns retain approximately 1 ml, so the final eluted volume will be 13 ml.

v) Rinse the pre-hybridised microarray slides in H2O for 1 minute, then in isopropanol for 1 minute. Spin at 1500 rpm for 5 minutes to dry slides. Keep in covered slide box. 1 NICK DORRELL - LAST UPDATE FEBRUARY 2004

vi) Prepare the Hybridisation solution as follows: - Sample 13 ml H2O 26 ml 20X SSC 12 ml 2% SDS 9 ml Heat at 95ºC for 2 minutes. Allow to cool slowly at room temperature and centrifuge for 30 seconds. Add 2 x 20 ml H2O to the corners of the hybridisation chamber. Place a slide into the chamber. Place a LifterSlip™ glass coverslip (22 mm x 25 mm) over the array section on the slide using tweezers. Pipette the Hybridisation solution onto the slide at the top of the coverslip. Seal the chamber and incubate in a water bath at 65°C overnight.

vii) Prepare Wash solutions as follows: - Wash A (1X SSC 0.5% SDS) Wash B (0.06X SSC) 20X SSC 20 ml 2.4 ml 20% SDS 1 ml H2O to 400 ml to 800 ml Incubate Wash A solution at 65ºC overnight. Dispense 400 ml volumes into three glass slide washing dishes. Remove slide(s) from the hybridisation chambers and gently remove coverslip(s) by rinsing in Wash A. Place slide(s) in a slide rack and rinse with agitation for 5 minutes. Transfer slide(s) to a clean slide rack and rinse with agitation in Wash B(i) for 2 minutes, then in Wash B (ii) for a further 2 minutes. Spin at 1500 rpm for 5 minutes to dry slide(s).

viii) Scan slide(s) using Affymetrix 418 scanner and analyse data

（NICK DORRELL - LAST UPDATE FEBRUARY 2004）

Monday, November 17, 2014

Protocol for Whole Animal/ Isolated Organ Vascular Perfusion Fixation( of rats/mice )

Advantages:

• Fixation begins immediately after arrest of systemic circulation. This minimizes the

alteration of cell structure resulting from post-mortem effects.

• Under in situ conditions, vascular perfusion results in a uniform and rapid

dissemination of fixative into all parts of the tissue via the vascular bed, resulting in an

increased depth and rate of actual fixation.

• The manipulation of tissues after the arrest of the systemic circulation but prior to

fixation is minimized resulting in many fewer artifacts.

• Many organs/ tissues may be effectively fixed at one time, thus maximizing the use of

each animal.

• In the case of immunocytochemical procedures employing relatively mild fixation

conditions, fewer autolytic artifacts result; redistribution or translocation of cellular

components is minimized; and greater immunocytochemical activity is retained.

Pre-perfusion:

1. The rat/mouse is retrieved from the animal quarters and brought upstairs to the

surgery room in a cage. The person removing the animal should sign and date the

animal inventory card ( yellow card ) in the out column.

2. The rat/mouse is weighed and injected with a mixture consisting of ketaset(75

mg/ml) + xylazine(5 mg/ml). The recommended injectable dose of this anesthetic is 1

ml/gm of body weight, IM. Allow 10 to 15 minutes for anesthesia to occur, indicated by

the loss of sensory/ reflex response, i.e. non- response to tail pinching, or paw

pinching. (Note: Metofane, as an inhaled anesthetic, may be used instead of the

ketaset/ xylazine mixture. In this case, assistance of another person is advised.)

3. Once anesthetized, and during the surgical procedures for whole animal or isolated

organ perfusion, care should be taken to prevent heat loss in the anesthetized

animal. They are quite prone to hypothermia.( use of a lamp is recommended )

4. People should consult Michael for the perfusion method for their particular needs. In

general, an isolated organ perfusion will yield the best results, but concern should be

taken to ensure maximum, most efficient use of the rat/mouse. For example, in the

case of an isolated perfusion of the heart/lungs, the rest of the animal might still be

suitable for biochemistry.5. In most instances, the rat will expire during the perfusion. However, in cases where

the animal survives or lingers, cardiac puncture is the appropriate means of sacrifice.

6. Upon completion of the procedure the carcass is wrapped in a surgical pad/ or

benchcoat, placed in a plastic bag, and returned to the animal quarters in the cage.

The carcass is placed in the freezer; and check that the animal inventory card ( yellow

card ) is signed off.

Recommended routes for vascular perfusion:

• Whole animal- descending aorta or vena cava.

• Central nervous system/ pituitary- aorta, via the left ventricle.

• Kidney- descending aorta, proximal to its distal bifurcation.

• Liver- portal vein

Perfusion Protocol

Perfusion pressure, in most instances, should be maintained between 60 and 100

mm Hg. Use a sphygmomanometer or gravity feed apparatus... we have both!!!

1. Generally, it is optimal to aerate/oxygenate the flush and fixation prior to beginning.

This can be maintained during the perfusion.

2. Flush the animal/organ first with 1XPBS containing 1% sodium nitrite, pH 7.4 for 30

seconds.

3. Follow up flush with perfusion of fixative for 5 minutes. Fixative contains:

3% formaldehyde (freshly prepared from paraformaldehyde);

1.5% glutaraldehyde

2.5% sucrose

contained in 100mM cacodylate, pH 7.4

200mls of fixative should be plenty for a mouse; 500 mls for a rat.

4. After 5 minutes of continuous perfusion, organs can be harvested; appropriately

dissected in fixative/buffer (100mM cacodylate, 2.5% sucrose, pH 7.4); and tissue

pieces allowed to fix an additional 1 hour.

5. Wash in 0.1M Cac/2.5% sucrose pH 7.4 3X 15' EA.6. Post-fix with Palade's OsO4 for one hour on ice, light tight, under hood.

5 ml Palade's 1% OsO4 = 1 ml Acetate-veronal stock

+ 1.25 ml 4% OsO4

+ 1 ml 0.1N HCl

+ 1.75 ml ddH2O

Acetate-veronal stock = 1.15 g NaAcetate Anhydrous

(J.T. Baker 1-3470)

+ 2.943 g NaBarbituate (Veronal)

labelled Barbital--(Sigma B-0500)

to 100 ml with ddH2O

7. Rinse 1X with Kellenberger, then incubate for 1 to 2 hrs. at RT. (or preferably

overnight)

10 ml Kellenberger = 2 ml Acetate Veronal Stock

+ 2.8 ml 0.1N HCl

+ 5.1 ml ddH2O

+ 0.05 g Uranyl Acetate

Check pH with paper before adding UA.

(Should be ~6)

8. One quick rinse in ddH2O, then one quick rinse in 50% ethanol.

9. Dehydrate with graded series of cold (4

C) ethanol (70, 95, 100); then three 10’

washes in fresh 100% ETOH at rm. temp; and then finally two 5’ exchanges with

propylene oxide (PO).

14. Place in 50% PO/50% Epon (can be old) overnight, uncovered under vacuum (or

hood).

10. Replace with fresh, 100% epon, and leave under vacuum for 2-6 hours.

11. Embed in fresh, 100% Epon. Put typed or pencil-written label in dummy capsules

with wooden stick, at least two capsules per sample. Pour tissue out of tube into

mincing dish. Place tissue in flat mold with small amount of Epon (to avoid curling)

with a wooden stick and place in 60o oven overnight.