Single-step assembly of double guide plasmid (pCas9-Duo) for gene-editing in Plasmodium

Abhinay Ramaprasad

Published: 2024-06-12 DOI: 10.17504/protocols.io.n2bvjn9d5gk5/v1

Abstract

This protocol describes a one-pot GoldenGate assembly of a new dual-guide targeting plasmid (pCas9-Duo) expressing two distinct guide RNAs in order to enhance the chances of a successful Cas9-mediated modification at a target region in Plasmodium falciparum . Designed as part of SHIFTiKO (frameshift-based trackable inducible knockout) system¹ (based on²).

Attachments

pCas9-Duo.gb

Steps

gRNA oligo design

Add the overhangs "ATTG" and "AAAC" to forward and reverse oligos of gRNA1, and "TTGG" and "TAAA" to gRNA2 respectively.

Double guide plasmid containing two gRNA expression cassetteswith distinct insertion sites.

Anneal gRNA oligos

Set up annealing reactions.$$

µ µ µ µ µ µ µ

Step 1 37°C 0h 30m 0s

Step 2 94°C 0h 5m 0s

Step 3 90°C to 25°C RAMP 5°C

Step 4 4°C Hold

Single-step GoldenGate assembly

Make 1:200 dilution mixture of annealed gRNA1 and gRNA2.

1µL gRNA1 annealed reaction

1µL gRNA2 annealed reaction

198µL

Set up assembly reaction

2µL pCas9 200ng/µL

1µL 1:200 diluted oligo

0.5µL

2µL

12µL

Step 1 37°C 0h 5m 0s

Step 2 16°C 0h 5m 0s

Repeat Step 1-2 for 6 cycles

Step 3 4°C Hold

Transform, plate and screen colonies

Add 2µL of assembly reaction to 15µL ultracompetent cells (like ).

Place On ice for 0h 20m 0s .

Heat shock at 42°C for 0h 0m 40s and spread transformed cells on .

10.

Pick colonies, miniprep-isolate plasmids and screen for gRNA1 and gRNA2 insertions by Sanger sequencing using (CAGGAAACAGCTATGAC) and (ACTGGCCGTCGTTTTAC), respectively.