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Table 1 Strategies for target gene modification in P. falciparum

From: Diverse target gene modifications in Plasmodium falciparum using Bxb1 integrase and an intronic attB

Method Mechanism Required elements Advantages Key limitations
Single or double crossover (with or without SLI) Homologous recombination between a targeting sequence on transfection plasmid and the gene of interest 1 or 2 large homology arms on the plasmid (i) Does not require expression of heterologous nucleases or enzymes;
(ii) The foundation of allelic replacement strategies in malaria research
(i) Depends on coincidental genome breaks near the target;
(ii) Cannot prevent gradual loss of the desired modification due to plasmid loop-out (avoided by SLI);
(iii) Requires months, usually with rounds of drug cycling to select for genomic integration (reduced with SLI)
Zinc-finger nuclease (ZFN) Engineered ZFN produces a double-stranded break, which is repaired by homologous recombination with plasmid (i) Custom engineered ZFN for each target site;
(ii) 2 homology arms for HDR, typically on a separate plasmid
(i) Rapid gene editing;
(ii) No sequence-specific restrictions such as PAM;
(iii) Longer track-record of use than CRISPR-Cas9, including clinical trials in humans
(i) Expensive;
(ii) Requires custom ZFN production for each target site;
(iii) Off-target nuclease activity may be comparable to CRISPR-Cas9
CRISPR-Cas9 sgRNA directs Cas9 nuclease to produce a double-stranded break, which is repaired by homologous recombination with plasmid (i) Cas9 nuclease;
(ii) sgRNA expressed under a U6 promoter or by heterologous T7 polymerase;
(iii) 2 homology arms for HDR, typically on a separate plasmid
(i) Rapid gene editing
(ii) Easy to implement in most labs
(i) Cleavage limited to sites adjacent to PAM sequence;
(ii) Requires careful consideration of on-target efficiency score for optimal design;
(iii) Off-target cleavage
Bxb1 integrase and intronic attB attB introduced into target gene intron (or via an engineered synthetic intron) undergoes specific recombination with attP element on transfection plasmid, facilitated by Bxb1 integrase (i) Intronic attB;
(ii) Bxb1 integrase;
(iii) Desired gene modification is placed distal to attP on plasmid
(i) Rapid gene editing;
(ii) Allows diverse modifications on a gene of interest;
(iii) Does not depend on HDR, so avoids cloning of AT-rich homology arms;
(iv) Little or no risk of off-target recombination;
(v) Permits larger target site insertions than CRISPR-Cas9
(i) Requires production of cloned parasite with intronic attB for each gene of interest;
(ii) Location of intronic attB within the target gene must be carefully selected to enable desired gene modifications