Part:BBa_K1431401:Design

One gRNA Sequence for HIV-1

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Method

We used a method derived from the method described in the paper by Feng Zhang^{[http://www.nature.com/nbt/journal/v31/n9/abs/nbt.2647.html ZhangFgRNA]}.

Conserved Sequence Analysis

We first extracted all conserved regions from the NIH HIV-1 Reference Genome. In this step, we found around 10 alternatives for the next process. Here all screening processes are done in a per-strain basis because of the high mutability of the HIV-1 virus.

Strip out sequences without PAM

Supplementary Table 1 - Base Percentage of HIV-1 Aligned Genome 730bp-752bp
	A %	G %	C %	T %	Empty %	Non Empty %	A(Corrected)	G(Corrected)	C(Corrected)	T(Corrected)
730	0	0	0	56.47	43.53	56.47	0.00%	0.00%	0.00%	100.00%
731	0	55.88	0	0.59	43.53	56.47	0.00%	98.96%	0.00%	1.04%
732	0	0	0	56.47	43.53	56.47	0.00%	0.00%	0.00%	100.00%
733	0	54.71	0	1.18	43.53	55.89	0.00%	97.89%	0.00%	2.11%
734	0	0	0	58.24	41.76	58.24	0.00%	0.00%	0.00%	100.00%
735	56.47	0.59	0.59	0.59	41.76	58.24	96.96%	1.01%	1.01%	1.01%
736	0	1.18	57.06	0	41.76	58.24	0.00%	2.03%	97.97%	0.00%
737	1.18	57.06	0	0.59	41.18	58.83	2.01%	96.99%	0.00%	1.00%
738	60	0	0	0	40	60	100.00%	0.00%	0.00%	0.00%
739	0.59	0	58.82	0	40	59.41	0.99%	0.00%	99.01%	0.00%
740	0	0	0	0	100	0
741	0	0	0	0	100	0
742	0.59	0	1.18	58.24	40	60.01	0.98%	0.00%	1.97%	97.05%
743	0	0	60	0	40	60	0.00%	0.00%	100.00%	0.00%
744	0	1.18	58.82	0	40	60	0.00%	1.97%	98.03%	0.00%
745	0	58.82	1.18	0	40	60	0.00%	98.03%	1.97%	0.00%
746	0.59	0	59.41	0	40	60	0.98%	0.00%	99.02%	0.00%
747	0.59	59.41	0	0	40	60	0.98%	99.02%	0.00%	0.00%
748	0.59	59.41	0	0	40	60	0.98%	99.02%	0.00%	0.00%
749	0	58.82	0.59	0.59	40	60	0.00%	98.03%	0.98%	0.98%
750	0.59	0.59	58.24	0.59	40	60.01	0.98%	0.98%	97.05%	0.98%
751	60	0	0	0	40	60	100.00%	0.00%	0.00%	0.00%
752	59.41	0.59	0	0	40	60	99.02%	0.98%	0.00%	0.00%

Select gRNA sequences with the best theoretical quality

HIV-1 Quasi-Conservative gRNAs(Useful)
Sequence	Rating(Zhang)	Rank(Church)	Free Energy(Approx.)
GTGTGGAAAATCTCTAGCAGTGG	71	-	-1.4	HIV1_REF_2010
TCTAGCAGTGGCGCCCGAACAGG	97	-	-1.3

Select gRNA sequences with the best theoretical quality

Source

Conserved Region of the HIV-1 Genome from the NIH HIV-1 Sequence Database