Haynes Lab:Notebook/CRISPR Editing/2014/08/07

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(08/07/2014)
Current revision (10:32, 8 August 2014) (view source)
(08/07/2014)
 
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| Target || Genomic DNA (template) || Primers || Amplicon (bp) || Expected S-Cut fragments
| Target || Genomic DNA (template) || Primers || Amplicon (bp) || Expected S-Cut fragments
|-
|-
-
| mCherry gRNA1d || CRISPR 1d || q1F / P3R || 329 || ~229, 100
+
| mCherry gRNA1 || CRISPR 1d || qPCR 1F / P3R || 329 || ~229, 100
|-
|-
-
| mCherry gRNA2d || CRISPR 2d || ### / ### || ### ||
+
| mCherry gRNA2 || CRISPR 2d || " || 329 || ~52, 277
 +
|-
 +
| mCherry gRNA4 || CRISPR 4d || " || 329 || ~180, 149
 +
|-
 +
| mCherry || mock || " || 329 || no cutting
 +
|-
 +
| luciferase gRNA20 || g20 +dox || gRNA 021 US P F / gRNA  027 ES P R || 1295 || ~165, 1130
 +
|-
 +
| luciferase gRNA20 || g20 -dox || " || 1295 || ~165, 1130
 +
|-
 +
| luciferase gRNA29 || g29 +dox || " || 1295 || ~395, 900
 +
|-
 +
| luciferase gRNA29 || g29 -dox || " || 1295 || ~395, 900
 +
|-
 +
| luciferase || mock +dox || " || 1295 || no cutting
 +
|-
 +
| luciferase || mock -dox || " || 1295 || no cutting
|}
|}
-
* Annotated map at https://benchling.com/cshlsynbio/mammalian-crispr/Rc6-kah154/edit
+
* mCherry annotated map at https://benchling.com/cshlsynbio/mammalian-crispr/Rc6-kah154/edit
 +
* Luciferase annotated map at https://benchling.com/rene/2014-cshl-synbio-mammalian-parts/FzI-puas-tk-luc-crispr-v-chromatin/edit
Line 73: Line 90:
| Sample || OD 260 || 260/280 || ng/uL
| Sample || OD 260 || 260/280 || ng/uL
|-
|-
-
| CRISPR "1" || --- || 1.82 || 40.3
+
| CRISPR 1d || 1.562 || 1.81 || 78.1
|-
|-
-
| CRISPR "2" || --- || 1.62 || 4.7
+
| CRISPR 2d || 0.273 || 1.71 || 14.0
|-
|-
-
| CRISPR "4" || --- || 1.85 || 9.8
+
| CRISPR 4d || 1.77 || 1.588 || 79.3
|-
|-
-
| Blank 1 || --- || 1.80 || 22.9
+
| g20 +dox || 1.408 || 1.79 || 70.4
|-
|-
-
| Blank 2 || --- || 1.54 || 5.0
+
| g20 -dox || 1.057 || 1.78 || 52.9
|-
|-
-
| Blank 3 || --- || 1.85|| 21.5
+
| g29 +dox || 0.987 || 1.77 || 49.4
 +
|-
 +
| g29 -dox || 0.856 || 1.82 || 42.8
 +
|-
 +
| +dox || 1.300 || 1.77 || 65.0
 +
|-
 +
| -dox || 1.169 || 1.8 || 58.5
 +
|-
 +
| U2OS mock || --- || --- || not done yet
|}
|}
-
* Combined Blank 1 and Blank 3, ~22 ng/μL in 100 uL volume
 
 +
'''Mismatch annealing''' (continued on 08/07/14)
 +
* Zhang Lab approach - do not mix with a wild type reference, since ~50% or more of the amplicons in the mutated cell sample will be wild type anyway
 +
** 200 ng clean PCR, 1x HF Phusion buffer, in final volume of 20 μL
-
'''Mismatch annealing'''
+
# CRISPR 1d
-
* Testing 2 methods
+
# CRISPR 2d
-
** IDT approach - mix the mutant PCR sample with a wild type reference sample to ensure heteroduplex formation
+
# CRISPR 4d
-
** Zhang Lab approach - do not mix with a wild type reference, since ~50% or more of the amplicons in the mutated cell sample will be wild type anyway
+
# g20 +dox
-
* 200 ng clean PCR, 1x HF Phusion buffer, in final volume of 15 μL
+
# g20 -dox
-
 
+
# g29 +dox
-
# 100 ng CRISPR "1" + 100 ng Blank (IDT approach)
+
# g29 -dox
-
# 200 ng CRISPR "1" (Zhang approach)
+
# +dox
-
# 200 ng Blank (wild type control)
+
# -dox
 +
# U2OS mock
Line 118: Line 146:
** 4 ºC/ hold ∞ 
** 4 ºC/ hold ∞ 
 +
 +
'''SURVEYOR nuclease reaction'''

Current revision

Project name Main project page
Previous entry      Next entry

08/07/2014

  • CRISPR on chromatin: transfections of g21, g22, g27 into Gal4EED/luc cells
  • Luc-mCh swap: building the donor sequence
  • SURVEYOR assay - C-on-C and mCherry



CRISPR on chromatin: transfections

  1. gRNA21/Cas9, dox+
  2. gRNA22/Cas9, dox+
  3. gRNA27/Cas9, dox+
  4. gRNA21/Cas9, dox-
  5. gRNA22/Cas9, dox-
  6. gRNA27/Cas9, dox-
  • On 8/05/14, cells were grown in 4 mL P/S-free medium to ~80% confluence in a 6-well plate, 3 with 1 μg/mL dox, 3 without dox



Luc-mCh swap: building the donor sequence

  • Designed nested PCR primers to gradually build homology arms onto mCherry. The resulting donor should...
    • Place mCherry in-frame upstream of the luciferase gene
    • Add an ATG start codon onto mCherry (we had to use KAH154 as a template, where mCherry has no start)
    • Add a double stop (TAATAG) onto the end of mCherry
    • Add 130 bp of homology with TK-luciferase to each end



SURVEYOR assay
Assistance from Week 2 Team 2


Planning

Target Genomic DNA (template) Primers Amplicon (bp) Expected S-Cut fragments
mCherry gRNA1 CRISPR 1d qPCR 1F / P3R 329 ~229, 100
mCherry gRNA2 CRISPR 2d " 329 ~52, 277
mCherry gRNA4 CRISPR 4d " 329 ~180, 149
mCherry mock " 329 no cutting
luciferase gRNA20 g20 +dox gRNA 021 US P F / gRNA 027 ES P R 1295 ~165, 1130
luciferase gRNA20 g20 -dox " 1295 ~165, 1130
luciferase gRNA29 g29 +dox " 1295 ~395, 900
luciferase gRNA29 g29 -dox " 1295 ~395, 900
luciferase mock +dox " 1295 no cutting
luciferase mock -dox " 1295 no cutting


PCR reaction

  • Reactions: 20 ng template DNA, 1x HF Phusion buffer, 0.2 mM dNTPs, 2.5 μL of 10 μM for each primer, 0.5 μL Phusion polymerase; in 50 μL final volume
  • Program
    • 98°C/ 30 sec
    • 30x [98°C/ 10 sec, 58°C/ 15 sec, 72°C/ 15 sec]
    • 72°C/ 10 min
    • 4°C hold


PCR product clean-up

  • Used QIAquick PCR Purification kit
  • Elute with 50 μL H2O
Sample OD 260 260/280 ng/uL
CRISPR 1d 1.562 1.81 78.1
CRISPR 2d 0.273 1.71 14.0
CRISPR 4d 1.77 1.588 79.3
g20 +dox 1.408 1.79 70.4
g20 -dox 1.057 1.78 52.9
g29 +dox 0.987 1.77 49.4
g29 -dox 0.856 1.82 42.8
+dox 1.300 1.77 65.0
-dox 1.169 1.8 58.5
U2OS mock --- --- not done yet


Mismatch annealing (continued on 08/07/14)

  • Zhang Lab approach - do not mix with a wild type reference, since ~50% or more of the amplicons in the mutated cell sample will be wild type anyway
    • 200 ng clean PCR, 1x HF Phusion buffer, in final volume of 20 μL
  1. CRISPR 1d
  2. CRISPR 2d
  3. CRISPR 4d
  4. g20 +dox
  5. g20 -dox
  6. g29 +dox
  7. g29 -dox
  8. +dox
  9. -dox
  10. U2OS mock


  • Thermal cycler program
    • 95ºC/ 10 min
    • 95ºC -- (‐2.0 ºC/s) --> 85ºC
    • 85ºC/ 1 min
    • 85ºC -- (‐0.3 ºC/s) --> 75ºC
    • 75ºC/ 1 min
    • 75ºC -- (‐0.3 ºC/s) --> 65ºC
    • 65ºC/ 1 min
    • 65ºC -- (‐0.3 ºC/s) --> 55ºC
    • 55ºC/ 1 min
    • 55ºC -- (‐0.3 ºC/s) --> 45ºC
    • 45ºC/ 1 min
    • 45ºC -- (‐0.3 ºC/s) --> 35ºC
    • 35ºC/ 1 min  
    • 35ºC -- (‐0.3 ºC/s) --> 25 ºC
    • 25ºC/ 1 min
    • 4 ºC/ hold ∞ 


SURVEYOR nuclease reaction



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