An integrated platform for genome engineering and gene expression perturbation in Plasmodium falciparum

Establishing robust genome engineering methods in the malarial parasite, Plasmodium falciparum, has the potential to substantially improve the efficiency with which we gain understanding of this pathogen’s biology to propel treatment and elimination efforts. Methods for manipulating gene expression and engineering the P. falciparum genome have been validated. However, a significant barrier to fully leveraging these advances is the difficulty associated with assembling the extremely high AT content DNA constructs required for modifying the P. falciparum genome. These are frequently unstable in commonly-used circular plasmids. We address this bottleneck by devising a DNA assembly framework leveraging the improved reliability with which large AT-rich regions can be efficiently manipulated in linear plasmids. This framework integrates several key functional genetics outcomes via CRISPR/Cas9 and other methods from a common, validated framework. Overall, this molecular toolkit enables P. falciparum genetics broadly and facilitates deeper interrogation of parasite genes involved in diverse biological processes.


pSwing construction
pJAZZ-OC Not I Vector (200 ng) was digested with NotI at 37 °C for 90 min and the reaction heat inactivated at 65 °C for 20 min. The pSwing gblock (Supplementary Methods Figure 2) containing the unique sequences 1, 3 and X (SEQ1, SEQ3, SEQX) and FseI, SacII and I-SceI restriction sites was obtained from IDT. The digested vector (20 ng) and gblock (20 ng) were mixed with an equal volume of 2x Gibson Assembly Master Mix and incubated at 50 ºC for 1 h to assemble an intermediate vector, pSwing. Big Easy TSA cells were transformed with 1 µL Gibson reaction mixture, and plated on LB-agar with chloramphenicol (34 µg/mL) and incubated overnight at 30 ºC. Selected colonies were grown overnight in liquid LB supplemented with chloramphenicol (34 µg/mL), mini-prepped and verified by restriction digestion and sequencing. were first modified to produce pSN372 and pSN1847. pSN372 was made by inserting via Gibson assembly: (1) a loxP-containing DNA fragment (tctattattaaataaatttaatggaataacttcgtatagcatacattatacgaagttatccggtttagccctcccacacataac; loxP site in bold font and Gibson overlap underlined) via an AgeI site upstream of BSD; and (2) a second loxP-containing fragment (atacctaatagaaatatatcttaagataacttcgtatagcatacattatacgaagttataataaatacctaatacaatccaggccaatcca ggcagagaaaggtcgatac; loxP site in bold, spacer in italics and Gibson overlap underlined) via an AflII site downstream of the Cam promoter. pSN1847 was created by performing the second step above on pMG56. Linear pSN372L and pSN1847L were created from their respective pSN372 and pSN1847 circular plasmids. pSwing (200 ng) was digested with SacII at 37 °C for 1.5 h, followed by heat inactivation of enzyme at 65 °C for 20 min. Inserts from pSN372 and pSN1847 were released by ScaI+NotI double digestion at 37 °C for 1 h, followed by heat inactivation of enzymes at 80 °C for 20 min. Released inserts (20 ng) were each mixed with SacII-digested pSwing (20 ng) and an equal volume 2x Gibson Assembly Master Mix, and incubated at 50 ºC for 1 h. Big Easy TSA cells were transformed and colonies analyzed as above to isolate pSN372L and pSN1847L (Supplementary Methods Figure 3A,B). Mini-prepped plasmids were analyzed for proper assembly by NotI and FseI+AflII (pSN372L) and Aflll+NotI (pSN1847L) digestions.
To obtain pSN054, I-SceI-digested pSN053 was Gibson assembled with pSN054-conversion gblock to yield pSN054. Thus, pSN054 differs from pSN053 in that the I-SceI site is immediately downstream of SEQ3 and SEQX is deleted (Supplementary Methods Figure 6E).

D. Assembling donor vectors to engineer the 3'-UTR of P. falciparum loci using pSN053/054 (Estimated time: 10 days)
Day 1: Installation of right homologous region (RHR) 1. Digest 200 ng pSN053/054 plasmid with I-SceI in a 10 µL at 37 ºC for 90 min, followed by heat inactivation of enzyme at 80 ºC for 20 min. 2. PCR the RHR from gDNA using a high-fidelity polymerase and gel purify the product.

E. Assembling donor vectors to achieve dual TetR aptamer-mediated target gene regulation at their native loci using pSN053/054 (Estimated time: 13 days)
Day 1: Installation of right homologous region (RHR) 1. Digest 200 ng pSN053/054 plasmid with I-SceI in 10 µL at 37 ºC for 90 min, followed by heat inactivation of enzyme at 80 ºC for 20 min. 2. PCR the RHR from gDNA using a high-fidelity polymerase and gel purify. Resuspend to 20 ng/µL. 3. Mix 20 ng each of RHR and digested vector with 3 µL 2x Gibson Assembly master mix and incubate at 50 ºC for 1 h. 4. Transform BigEasy-TSA Electrocompetent Cells with 1 µL Gibson reaction mixture.

Day 5
Pick 8 colonies and grow overnight at 30 ºC in liquid LB media containing chloramphenicol (34 µg/mL) and 0.2% w/v arabinose.   Figure 5C. (F-I) Uncropped blots corresponding to those presented in Figure 5D but merged with the molecular weight marker detected using colorimetric imaging. The blots were initially probed with mouse anti-HA antibody (top panels), re-blocked, and probed with rabbit anti-GAPDH (bottom panels). Short and long membrane exposure conditions were carried out for optimal detection and presentation of CK and CRT signals.  Figure 7B. (D-E) Uncropped blots corresponding to those presented in Figure 7C, but merged with the molecular weight marker detected using colorimetric imaging. The blots were initially probed with mouse anti-HA antibody (top panels), re-blocked, and probed with rabbit anti-GAPDH (bottom panels).