TITLE: In vitro testing of guide RNA efficiency for CRISPR-mediated genome editing
AUTHORS: Srinidhi Holalu, Angela Fang, Benjamin Blackman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>In CRISPR-mediated genome editing, guide RNAs (gRNAs) serve to target the Cas9 nuclease to an intended target genomic locus. Many gRNAs are not efficient in facilitating Cas9-mediated cleavage by introduction of double-strand breaks. Therefore, after designing possible gRNA sequences via web-based tools, it is critical to assess their cleavage efficiency at the target locus and identify the most efficient gRNA(s) </span><span style = "font-style:italic;">in vitro</span><span> before deploying in binary plasmids for genome editing </span><span style = "font-style:italic;">in planta</span><span style = "font-style:italic;">.</span></div></div>

[STEPS]
?. [Cloning a template fragment from genomic DNA]
Perform DNA extraction using CTAB method or any other methods that you may use routinely for your plant species.
?. [Cloning a template fragment from genomic DNA]
Design primers flanking your target PAM sites (NGG). PCR to amplify around 500-600 bp of genomic/gene fragment around the target. Large size templates are easy to resolve on gel electrophoresis.
?. [Cloning a template fragment from genomic DNA]
Clean PCR product using Zymo/Bioneer DNA cleanup kit and Sanger sequence to confirm that your PCR amplifications are intended gene. This will also help later for screening indel polymorphisms at PAM site.
?. [Designing gRNA and synthesis of guide RNA using in vitro T7 kit: Oligo Design]
Design targets for editing target exon(s) using gRNA design tool (http://crispr.mit.edu/ or any other portal). The following primers are then used to synthesize the DNA template to prepare gRNA for each target.Oligo design a.   Primer 1 (specific for each guide): a) TAATACGACTCCTATAGGG-(20 bp guide sequence)-gtttAagagctaTGCTGgaa b) The three bolded G’s are included in the transcript and required for transcription. If the guide-specific sequence starts with one or more G’s, the same number of bolded G’s can be deleted from the primer.b. Primer 2 (always the same): a) Reverse-gRNA Template: aaaagcaccgactcggtgccactttttcaagttgataacggactagccttatttAaacttgctaTGCTGtttc CAGCAtagctctTaaac
?. [gRNA synthesis]
Set up reaction in PCR tube: water (nuclease free)  NTP mix  each  Template DNA (∼  — ) T7 RNA Polymerase Mix Total
7 µl
5 µl
2 µl
100 ng
500 ng
1 µl
15 µl
?. [gRNA synthesis]
Use HiScribe T7 High Yield RNA Synthesis Kit (NEB E2050S)
?. [gRNA synthesis]
Incubate at  for .
37 °C
?. [gRNA synthesis]
Move the reaction into  eppendorf tube.
1.5 ml
?. [gRNA synthesis]
Add  DNAase/RNAse free water.
435 µl
?. [gRNA synthesis]
Measure gRNA concentration on nanodrop. Note it down. (May not be accurate, as NTPs may inflate the nanodrop readings).
?. [gRNA synthesis]
Optional to cleanup using any RNA clean-up kit. Dilute to  by adding nuclease free water.
?. [Designing gRNA and synthesis of guide RNA using in vitro T7 kit: PCR Double-stranded DNA synthesis for gRNA]
Set up the following PCR reaction: nuclease free water  5X Phusion buffer  dNTP  primer 1 ()  primer 2 () Always Primer 2 (Reverse-gRNA Template) Phusion polymeraseTotal
30.5 µl
10 µl
1 µl
4 µl
4 µl
0.5 µl
50 µl
?. [Designing gRNA and synthesis of guide RNA using in vitro T7 kit: PCR Double-stranded DNA synthesis for gRNA]
Program for PCR:1. 2. 3. 8X-10X cycles of these three steps 4. 5. 6.  hold
98 °C
98 °C
55 °C
72 °C
72 °C
4 °C
?. [Designing gRNA and synthesis of guide RNA using in vitro T7 kit: PCR Double-stranded DNA synthesis for gRNA]
Nano-drop to quantify the PCR product. Run a small aliquot of the PCR product out on a gel to check the bands. The resulting band should be 90 bp (very small) and so it is necessary to run a ≥ 3 % agarose gel.
?. [Designing gRNA and synthesis of guide RNA using in vitro T7 kit: PCR Double-stranded DNA synthesis for gRNA]
Purify the PCR product using Zymo/bioneer DNA cleanup kit.
?. [Cas9 nuclease assay and gel electrophoresis: Incubation of template DNA with gRNA and Cas9 nuclease (NEB Cas9 M0386S)]
Set up reaction (include a negative RNA control): water (nuclease free)  10X Cas9 reaction buffer  sgRNA ()  Cas9 Nuclease Total
7.5 µl
1.5 µl
1.5 µl
0.5 µl
11 µl
?. [Cas9 nuclease assay and gel electrophoresis: Incubation of template DNA with gRNA and Cas9 nuclease (NEB Cas9 M0386S)]
Pre-incubate for  at .
37 °C
?. [Cas9 nuclease assay and gel electrophoresis: Incubation of template DNA with gRNA and Cas9 nuclease (NEB Cas9 M0386S)]
Add  substrate DNA (∼  —  total).
4 µl
50 ng
100 ng
?. [Cas9 nuclease assay and gel electrophoresis: Incubation of template DNA with gRNA and Cas9 nuclease (NEB Cas9 M0386S)]
Incubate  at  (or longer up to ).
37 °C
?. [Cas9 nuclease assay and gel electrophoresis: Incubation of template DNA with gRNA and Cas9 nuclease (NEB Cas9 M0386S)]
Denature Cas9 by incubation at  for .
80 °C
?. [Cas9 nuclease assay and gel electrophoresis: Incubation of template DNA with gRNA and Cas9 nuclease (NEB Cas9 M0386S)]
Set up a control reaction including all the above ingredients without gRNA. In addition, different ratios of gRNA to Cas9 nuclease can also be setup to replicate the efficiency tests.
?. [Cas9 nuclease assay and gel electrophoresis: Gel Electrophoresis]
Prepare  agarose gel.
?. [Cas9 nuclease assay and gel electrophoresis: Gel Electrophoresis]
Mix the Cas9 incubated reaction with  of loading dye. Load the mixture on gel. If stain was not added during gel preparation, soak gel in staining buffer.
2 µl
?. [Cas9 nuclease assay and gel electrophoresis: Gel Electrophoresis]
Run gel electrophoresis until the ladder resolves and check the cleavage of template DNA under UV illumination.